Anti-system asc amino acid transporter 2 (asct2) antibody

ABSTRACT

An object of the present invention is to provide a monoclonal antibody which is useful for treating or diagnosing a disease relating to system ASC amino acid transporter 2 (hereinafter, referred to as “ASCT2”) or a method using the antibody. The present invention provides a monoclonal antibody which specifically recognizes a native three-dimensional structure of an extracellular region of ASCT2 and binds to the extracellular region, or an antibody fragment thereof; a hybridoma which produces the antibody; a DNA which encodes the antibody; a vector which contains the DNA; a transformant obtainable by introducing the vector; a process for producing an antibody or an antibody fragment thereof using the hybridoma or the transformant; and a therapeutic agent using the antibody or the antibody fragment thereof, and a diagnostic agent using the antibody or the antibody fragment thereof.

BACKGROUND OF ME INVENTION

1. Field of the Invention

The present invention relates to a monoclonal antibody whichspecifically recognizes a native three-dimensional structure of anextracellular region of system ASC amino acid transporter 2(hereinafter, referred to as “ASCT2”) and binds to the extracellularregion, or an antibody fragment thereof; a hybridoma which produces theantibody; a DNA which encodes the antibody; a vector which contains theDNA; a transformant obtainable by introducing the vector; a process forproducing an antibody or an antibody fragment thereof using thehybridoma or the transformant; and a therapeutic agent using theantibody or the antibody fragment thereof, and a diagnostic agent usingthe antibody or the antibody fragment thereof.

2. Brief Description of the Background Art

An ASCT2 polypeptide is a 10-times transmembrane protein consisting of541 amino acids in full-length form, and functions as a transporterwhich transports neutral amino acids through a cell membrane dependingon a sodium ion. The amino acid transporter is categorized into severalsystems, based on functional characteristics including substratespecificity and the like. ASCT2 belongs to System ASC and has functionsof intracellular uptake of neutral amino acids such as L-alanine,L-serine, L-threonine, L-cysteine, and L-glutamine depending on a sodiumion (Non-Patent Document 1).

Further, ASCT2 is a viral cell-surface receptor which is shared by typeD simian retrovirus and three type C viruses [feline endogenous virus(RD114), baboon endogenous virus, and avian reticuloendotheliosis virus](Non-Patent Documents 2 and 3).

ASCT2 is also known as sodium-dependent neutral amino acid transportertype 2, adipocyte amino acid transporter, AAAT, neutral amino acidtransporter B, MB, amino acid transporter B⁰, MB⁰, ATBO, baboon M7 virusreceptor, M7V1, M7VS1, insulin-activated amino acid transporter,FLJ31068, RD 114 virus receptor, RD114/simian type D retrovirusreceptor, RDR, RDRC, or R16 (Non-patent Documents 1 and 3).

Furthermore, as another name, ASCT2 is known as Solute carrier family 1(neutral amino acid transport), member 5, Solute carrier family 1 member5, or SLC1A5 and belongs to SLC1 family.

With respect to the relationship between the development of cancer andthe expression of ASCT2 or SLC1A5, it has been found that expressionlevels of three SLC1A5, SLC7A5 and SLC38A5 are significantly increasedin cancerous tissues by studies using expressed sequence tag (EST)database (Non-Patent Document 4). Further, the expression of SLC1A5 isnot recognized in a normal liver, but is recognized in clinical tissuesof hepatocellular carcinoma or hepatoblastoma (Non-Patent Document 5).In addition, it has been found that the expression of SLC1A5 is higherin clinical tissues of poorly-differenciated astrocytoma or glioblastomamultiforme than that of normal tissues (Non-Patent Document 6).

Further, the expression of ASCT2 is detected in clinical tissues ofcolorectal cancer and prostate cancer by immunohistological staining orWestern blotting, and patients who highly express ASCT2 have a pooroutcome (Non-Patent Documents 7 and 8).

Furthermore, ASCT2 is responsible for the uptake of glutamine in severalcell lines of colorectal cancer, liver cancer, breast cancer,astrocytoma and neuroblastoma (Non-Patent Documents 9 to 12), and theproliferation of cells is suppressed by competitively inhibitingintracellular uptake of glutamine using an alanine-serine-threoninemixture which is a substrate for ASCT2 (Non-Patent Document 9).

As regards an antibody which binds to ASCT2, a polyclonal antibodyagainst partial peptides of the intracellular N-terminal or C-terminalof human ASCT2 is known (Non-Patent Documents 13, 14 and 15). Since allof these are antibodies which recognize an intracellular portion ofASCT2, they cannot bind to ASCT2 which is expressed on the cell membraneof cells. It is known that when an antibody binds to an antigen proteinexpressed on the cell membrane, a cellular cytotoxicity due to aneffector activity of the antibody, such as antibody-dependent cellularcytotoxicity (hereinafter, referred to as “ADCC activity”) orcomplement-dependent cytotoxicity (hereinafter, referred to as “CDCactivity”) is induced within the living body (Non-Patent Document 16).However, since these antibodies cannot bind to ASCT2 expressed on thecell membrane of cells, these cellular cytotoxicities due to theeffector activity cannot be expected. Further, since all of theseantibodies are antibodies which recognize an intracellular portion ofASCT2, they cannot inhibit intracellular uptake of amino acids by ASCT2which expresses in living cells.

In addition, polyclonal antibodies which recognize human ASCT2 arecommercially available from LifeSpan BioSciences (Catalog Numbers:LS-C16840 and LC-C31887), Avia Systems Biology (Catalog Number:ARP42247_T100), Santa Cruz Biotechnology (Catalog Numbers: sc-50698 andsc-50701), or Millipore (Catalog Number: AB5468).

However, it is not known a monoclonal antibody which specificallyrecognizes a native three-dimensional structure of an extracellularregion of ASCT2 and binds to the extracellular region.

-   [Non-Patent Document 1] J. Biol. Chemistry, 271, 18657 (1996)-   [Non-Patent Document 2] Proc. Natl. Acad. Sci. USA, 96, 2129 (1999)-   [Non-Patent Document 3] J. Virology, 73, 4470 (1999)-   [Non-Patent Document 4] Seminars in Cancer Biology, 15, 254 (2005)-   [Non-Patent Document 5] Am. J. Physiol. Gastrointest. Liver    Physiol., 283, G1062 (2002)-   [Non-Patent Document 6] NeuroReport, 15, 575 (2004)-   [Non-Patent Document 7] Anticancer Research, 22, 2555 (2002)-   [Non-Patent Document 8] Anticancer Research, 23, 3413 (2003)-   [Non-Patent Document 9] J. Surgical Research, 90, 149 (2000)-   [Non-Patent Document 10] J. Cellular Physiology, 176, 166 (1998)-   [Non-Patent Document 11] J. Neuroscience Research, 66, 959 (2001)-   [Non-Patent Document 12] Am. J. Physiol. Cell Physiol., 282, C1246    (2002)-   [Non-Patent Document 13] J. Gene Medicine, 6, 249 (2004)-   [Non-Patent Document 14] Am. J. Physiol. Cell Physiol., 281, C963    (2001)-   [Non-Patent Document 15] Biochem. J., 382, 27 (2004)-   [Non-Patent Document 16] Cancer Res., 56, 1118 (1996)

SUMMARY OF THE INVENTION

An object of the present invention is to provide a monoclonal antibodywhich specifically recognizes a native three-dimensional structure of anextracellular region of ASCT2 and binds to the extracellular region, oran antibody fragment thereof a hybridoma which produces the antibody; aDNA which encodes the antibody; a vector which contains the DNA; atransformant obtainable by introducing the vector; a process forproducing an antibody or an antibody fragment thereof using thehybridoma or the transformant; and a therapeutic agent using theantibody or the antibody fragment thereof, and a diagnostic agent usingthe antibody or the antibody fragment thereof.

The present invention can provide a monoclonal antibody whichspecifically recognizes a native three-dimensional structure of anextracellular region of ASCT2 and binds to the extracellular region, oran antibody fragment thereof; a hybridoma which produces the antibody; aDNA which encodes the antibody; a vector which contains the DNA; atransformant obtainable by introducing the vector; a process forproducing an antibody or an antibody fragment thereof using thehybridoma or the transformant; and a therapeutic agent using theantibody or the antibody fragment thereof, and a diagnostic agent usingthe antibody or the antibody fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(1) shows the alignment of NM_(—)005628 and HCHON2001712.*represents a site where both nucleotide sequences are same.

FIG. 1(2) shows the alignment of NM_(—)005628 and HCHON2001712.*represents a site where both nucleotide sequences are same.

FIG. 1(3) shows the alignment of NM_(—)005628 and HCHON2001712.*represents a site where both nucleotide sequences are same. The designpositions of amplification primers used in Q-PCR are underlined (Fw#1corresponds to nucleotides at positions 2281 to 2301 of NM_(—)005628 andnucleotides at positions 1689 to 1709 of HCHON2001712, and Rv#1corresponds to a reverse chain of nucleotides at positions 2739 to 2719of NM_(—)005628 and a reverse chain of nucleotides at positions 2139 to2119 of HCHON2001712).

FIG. 1(4) shows the alignment of MN_(—)005628 and HCHON2001712.*represents a site where both nucleotide sequences are same. The designpositions of amplification primers used in Q-PCR are underlined (Fw#1corresponds to nucleotides at positions 2281 to 2301 of NM_(—)005628 andnucleotides at positions 1689 to 1709 of HCHON2001712, and Rv#1corresponds to a reverse chain of nucleotides at positions 2739 to 2719of NM_(—)005628 and a reverse chain of nucleotides at positions 2139 to2119 of HCHON2001712).

FIG. 2(1) shows expression levels of mRNA of ASCT2 gene in cancer celllines, xenografts, normal tissues and clinical cancer tissues.

FIG. 2(2) shows expression levels of mRNA of ASCT2 gene in cancer celllines, xenografts, normal tissues and clinical cancer tissues.

FIG. 3 shows the reactivity of an anti-myc antibody (Anti-Myc) and ananti-H is antibody (Anti-His) to immobilized human ASCT2-myc/Hisgene-introduced CHO cells (ASCT2/CHO) and vector-introduced CHO cells(Vector/CHO), as measured by a flow cytometer. The abscissa representsthe fluorescence intensity, and the ordinate represents the cell counts.The antibody is indicated by a black curve, and buffer is indicated by awhite curve.

FIG. 4 shows animal species and antibody classes of anti-ASCT2monoclonal antibodies obtained in Examples.

FIG. 5 shows the reactivity of a monoclonal antibody KM43842 against theN-terminal peptide of ASCT2 to an N-terminal peptide of ASCT2, inbinding ELISA. The abscissa represents an antibody concentration(μg/mL), and the ordinate represents an absorbance ratio (OD415/490). ▴represents an absorbance ratio in the case of using a human ASCT2N-terminal peptide-THY conjugate and ▪ represents an absorbance ratio inthe case of using a control peptide-THY conjugate.

FIG. 6 shows the reactivity of an anti-ASCT2 monoclonal antibody KM3998to ASCT2-myc/His gene-introduced CHO cells (ASCT2/CHO),vector-introduced CHO cells (Vector/CHO), and KMS-11 by means offluorescent cell staining (flow cytometer). The abscissa represents thefluorescence intensity, and the ordinate represents the cell counts.KM3998 is indicated by a black curve, and rat IgG2a-UNLB is indicated bya white curve.

FIG. 7 shows the reactivity of anti-ASCT2 monoclonal antibodies KM4000,KM4001, KM4008, KM4012 and KM4018 to human ASCT2-myc/His gene-introducedCHO cells (ASCT2/CHO), vector-introduced CHO cells (Vector/CHO) andKMS-11 by means of fluorescent cell staining (flow cytometer). Theabscissa represents an antibody concentration (μg/mL), and the ordinaterepresents a mean fluorescence intensity. The mean fluorescenceintensity is indicated by  and solid line for KM511, by ▪ and thicksolid line for KM4000, by ▴ and dotted line for KM4001, by ◯ and dottedline for KM4008, by □ and dotted line for KM4012, by the symbol □ andsolid line for rat IgG2a-UNLB, and by ▴ and thick solid line for KM4018.

FIG. 8 shows the inhibitory activity of an anti-ASCT2 monoclonalantibody KM3998 on glutamine-dependent proliferation of the humancolorectal cancer cell line WiDr. The abscissa represents a finalconcentration (μg/mL) of KM3998, and the ordinate represents a relativevalue (%) obtained by taking proliferation of non-antibody treated cellsto be 100%. The relative proliferation rate is indicated by ♦ and solidline for KM3998, by x and solid line for rat IgG2a-UNLB, and by dottedline for AST mixture.

FIG. 9 shows the inhibitory activities of anti-ASCT2 monoclonalantibodies KM4000, KM4001, KM4008, KM4012 and KM4018 onglutamine-dependent proliferation of the human colorectal cancer cellline WiDr. The abscissa represents a final concentration (μg/mL) ofindividual antibodies, and the ordinate represents a relative value (%)obtained by taking proliferation of non-antibody treated cells to be100%. The relative proliferation rate is indicated by ♦ and solid linefor KM4000, by ▪ and solid line for KM4001, by Δ and solid line forKM4008, by ◯ and solid line for KM4012, by x and solid line for KM511,by ▪ and solid line for KM4018, by x and solid line for rat IgG2a-UNLB,and by dotted line for AST mixture.

FIG. 10 shows the reactivity of an anti-ASCT2 human chimeric antibody tohuman ASCT2-myc/His gene-introduced CHO cells (ASCT2/CHO) and humanmultiple myeloma cell line OPM-2 by means of fluorescent cell staining(flow cytometer). The abscissa represents an antibody concentration(μg/mL), and the ordinate represents a mean fluorescence intensity. Themean fluorescence intensity is indicated by ◯ and solid line forcKM4008, by Δ and solid line for cKM4012, and by ▪ and solid line forcKM4018.

FIG. 11 shows the ADCC activity of an anti-ASCT2 human chimeric antibodyon the human multiple myeloma cell line KMS-11. The abscissa representsan antibody concentration (ng/mL), and the ordinate represents an ADCCactivity. The ADCC activity is indicated by ◯ and solid line forcKM4008, by Δ and solid line for cKM4012, and by ▪ and solid line forcKM4018.

FIG. 12 shows the CDC activity of an anti-ASCT2 human chimeric antibodyon human ASCT2-myc/His gene-introduced CHO cells (ASCT2/CHO) and humancolorectal cancer cell line Colo205. The abscissa represents an antibodyconcentration (μg/mL), and the ordinate represents a CDC activity. TheCDC activity is indicated by ◯ and solid line for cKM4008, by Δ andsolid line for cKM4012, and by ▪ and solid line for cKM4018.

FIG. 13 shows the inhibitory activity of anti-ASCT2 human chimericantibodies on glutamine-dependent proliferation of the human colorectalcancer cell line WiDr. The abscissa represents a final concentration(μg/mL) of individual antibodies, and the ordinate represents a relativevalue (%) obtained by taking proliferation of non-antibody treated cellsto be 100%. The relative proliferation rate is indicated by ◯ and solidline for cKM4008, by Δ and solid line for KM4012, by ▪ and solid linefor KM4018, by + and solid line for KM511 and by dotted line for ASTmixture.

FIG. 14 shows a schematic illustration of a human ASCT2 protein and amouse ASCT2 protein. Black color represents amino acids which aredifferent between the human and mouse proteins in putative extracellularregions (EL1, EL2, EL3, EL4 and EL5).

FIG. 15 shows the reactivity of anti-ASCT2 monoclonal antibodies KM4008,KM4012 and KM4018 with mouse ASCT2-myc/His gene-introduced CHO cells(mouse ASCT2/CHO) and mouse/human chimeric ASCT2/CHO [mouse EL1 typeASCT2-myc/His gene-introduced CHO cell line (mEL1/CHO), mouse EL2 typeASCT2-myc/His gene-introduced CHO cell line (mEL2/CHO), mouse EL3 typeASCT2-myc/His gene-introduced CHO cell line (mEL3/CHO), and mouse EL4type ASCT2-myc/His gene-introduced CHO cell line (mEL4/CHO)]. Thesymbols “+”, “−”, and “±” represent the presence of reactivity, theabsence of reactivity, and the decrease in reactivity, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the following (1) to (44):

[1] A monoclonal antibody which specifically recognizes a nativethree-dimensional structure of an extracellular region of system ASCamino acid transporter 2 (hereinafter, referred to as “ASCT2”) and bindsto the extracellular region, or an antibody fragment thereof;[2] The monoclonal antibody or the antibody fragment thereof describedin [1], which inhibits intracellular uptake of an amino acid throughASCT2;[3] The monoclonal antibody or the antibody fragment thereof describedin [1] or [2], which has cellular cytotoxicity;[4] The monoclonal antibody or the antibody fragment thereof describedin [3], in which the cellular cytotoxicity is an antibody-dependentcellular cytotoxicity (ADCC) activity or complement-dependentcytotoxicity (CDC) activity;[5] The monoclonal antibody or the antibody fragment thereof describedin any one of [1] to [4], which binds to human ASCT2 and does not bindto mouse ASCT2;[6] The monoclonal antibody or the antibody fragment thereof describedin [5], which binds to at least EL2 region of human ASCT2;[7] The monoclonal antibody or the antibody fragment thereof describedin [6], which binds to at least any one of amino acids selected from atpositions 154, 159 to 160, 163 to 171, 173 to 174, 177, 188, 204 to 205,207, 210 to 212, and 214 to 223 in the amino acid sequence representedby SEQ ID NO:2;[8] The antibody or the antibody fragment thereof described in any oneof [1] to [7], in which the monoclonal antibody is a monoclonal antibodywhich binds to an epitope which is recognized by a monoclonal antibodyproduced from any one of hybridomas selected from a hybridoma KM4008(FERM BP-10962) and a hybridoma KM4012 (FERM BP-10963);[9] The antibody or the antibody fragment thereof described in any oneof [1] to [8], in which the monoclonal antibody is a monoclonal antibodyproduced from any one of hybridomas selected from a hybridoma KM4008(FERM BP-10962) and a hybridoma KM4012 (FERM BP-10963);[10] The antibody fragment described in any one of [1] to [9], in whichthe antibody fragment is an antibody fragment selected from Fab, Fab′,F(ab′)₂, a single chain antibody (scFv), a dimerized V region (diabody),a disulfide stabilized V region (dsFv) and a CDR-containing peptide.[11] The antibody or the antibody fragment thereof described in any oneof [1] to [10], in which the monoclonal antibody is a recombinantantibody;[12] The recombinant antibody or the antibody fragment thereof describedin [11], in which the recombinant antibody is a recombinant antibodyselected from a human chimeric antibody, a humanized antibody and ahuman antibody;[13] The recombinant antibody or the antibody fragment thereof describedin [11], which comprises complementarity determining regions(hereinafter, referred to as “CDRs”) of a heavy chain variable region(hereinafter, referred to as “VH”) and a light chain variable region(hereinafter, referred to as “VL”) of the monoclonal antibody describedin any one of [1] to [9];[14] The recombinant antibody or the antibody fragment thereof describedin [13], in which CDR1, CDR2 and CDR3 of VH of the antibody comprise theamino acid sequences represented by SEQ ID NOs:26, 27 and 28,respectively;[15] The recombinant antibody or the antibody fragment thereof describedin [13], in which CDR1, CDR2 and CDR3 of VL of the antibody comprise theamino acid sequences represented by SEQ ID NOs:29, 30 and 31,respectively;[16] The recombinant antibody or the antibody fragment thereof describedin [13], in which CDR1, CDR2 and CDR3 of VII of the antibody comprisethe amino acid sequences represented by SEQ ID NOs:26, 27 and 28,respectively, and CDR1, CDR2 and CDR3 of VL of the antibody comprise theamino acid sequences represented by SEQ ID NOs:29, 30 and 31,respectively;[17] The recombinant antibody or the antibody fragment thereof describedin [13], in which CDR1, CDR2 and CDR3 of VH of the antibody comprise theamino acid sequences represented by SEQ ID NOs:32, 33 and 34,respectively;[18] The recombinant antibody or the antibody fragment thereof describedin [13], in which CDR1, CDR2 and CDR3 of VL of the antibody comprise theamino acid sequences represented by SEQ ID NOs:35, 36 and 37,respectively;[19] The recombinant antibody or the antibody fragment thereof describedin [13], in which CDR1, CDR2 and CDR3 of VH of the antibody comprise theamino acid sequences represented by SEQ ID NOs:32, 33 and 34,respectively, and CDR1, CDR2 and CDR3 of VL of the antibody comprise theamino acid sequences represented by SEQ ID NOs:35, 36 and 37,respectively;[20] The recombinant antibody or the antibody fragment thereof describedin [13], in which CDR1, CDR2 and CDR3 of VH of the antibody comprisecomprise the amino acid sequences represented by SEQ ID NOs:49, 50 and51, respectively,[21] The recombinant antibody or the antibody fragment thereof describedin [13], in which CDR1, CDR2 and CDR3 of VL of the antibody comprise theamino acid sequences represented by SEQ ID NOs:52, 53 and 54,respectively;[22] The recombinant antibody or the antibody fragment thereof describedin [13], in which CDR1, CDR2 and CDR3 of VH of the antibody comprise theamino acid sequences represented by SEQ ID NOs:49, 50 and 51,respectively, and CDR1, CDR2 and CDR3 of VL of the antibody comprise theamino acid sequences represented by SEQ ID NOs:52, 53 and 54,respectively;[23] The human chimeric antibody or the antibody fragment thereofdescribed in [12], which comprises VH and VL of the monoclonal antibodydescribed in any one of [1] to [8];[24] The human chimeric antibody or the antibody fragment thereofdescribed in [23], which is selected from a human chimeric antibody inwhich VH of the antibody comprises the amino acid sequence representedby SEQ ID NO:19 and VL of the antibody comprises the amino acid sequencerepresented by SEQ ID NO:21, a human chimeric antibody in which VH ofthe antibody comprises the amino acid sequence represented by SEQ IDNO:23 and VL of the antibody comprises the amino acid sequencerepresented by SEQ ID NO:25, and a human chimeric antibody in which VHof the antibody comprises the amino acid sequence represented by SEQ IDNO:46 and VL of the antibody comprises the amino acid sequencerepresented by SEQ ID NO:48;[25] The humanized antibody or the antibody fragment thereof describedin [12], in which VH of the antibody comprises the amino acid sequencerepresented by SEQ ID NO:71, or an amino acid sequence in which at leastone amino acid residue selected from Val at position 2, Ser at position9, Val at position 20, Ser at position 30, Arg at position 38, Glu atposition 46, Leu at position 86, Val at position 93, Tyr at position 95and Val at position 116 in the amino acid sequence represented by SEQ IDNO:71 is substituted with other amino acid residue, and/or in which VLof the antibody comprises an amino acid sequence represented by SEQ IDNO:72, or an amino acid sequence in which at least one amino acidresidue selected from Pro at position 8, Val at position 15, Gln atposition 38, Ala at position 43, Pro at position 44, Phe at position 71and Tyr at position 87 in the amino acid sequence represented by SEQ IDNO:72 is substituted with other amino acid residue;[26] The humanized antibody or the antibody fragment thereof describedin the above [25], in which VH of the antibody comprises an amino acidsequence in which at least one modification selected from modificationsfor substituting Val at position 2 with Ile, Ser at position 9 with Pro,Val at position 20 with Ile, Ser at position 30 with Thr, Arg atposition 38 with Lys, Glu at position 46 with Lys, Leu at position 86with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, andVal at position 116 with Leu is introduced in the amino acid sequencerepresented by SEQ ID NO:71, and VL of the antibody comprises an aminoacid sequence in which at least one modification selected frommodifications for substituting Pro at position 8 with Thr, Val atposition 15 with Leu, Gln at position 38 with Arg, Ala at position 43with Thr, Pro at position 44 with Val, Phe at position 71 with Tyr, andTyr at position 87 with Phe is introduced in the amino acid sequencerepresented by SEQ ID NO:72;[27] The humanized antibody or the antibody fragment thereof describedin [25], in which a humanized antibody is selected from a humanizedantibody in which VH of the antibody comprises an amino acid sequencerepresented by SEQ ID NO:76 and/or VL of the antibody comprises an aminoacid sequence represented by SEQ ID NO:72, a humanized antibody in whichVH of the antibody comprises an amino acid sequence represented by SEQID NO:78 and/or VL of the antibody comprises an amino acid sequencerepresented by SEQ ID NO:72, a humanized antibody in which VH of theantibody comprises an amino acid sequence represented by SEQ ID NO:80and/or VL of the antibody comprises an amino acid sequence representedby SEQ ID NO:72, a humanized antibody in which VH of the antibodycomprises an amino acid sequence represented by SEQ ID NO:82 and/or VLof the antibody comprises an amino acid sequence represented by SEQ IDNO:72, a humanized antibody in which VH of the antibody comprises anamino acid sequence represented by SEQ ID NO:71 and/or VL of theantibody comprises an amino acid sequence represented by SEQ ID NO:84, ahumanized antibody in which VH of the antibody comprises an amino acidsequence represented by SEQ ID NO:76 and/or VL of the antibody comprisesan amino acid sequence represented by SEQ ID NO:84, a humanized antibodyin which VH of the antibody comprises an amino acid sequence representedby SEQ ID NO:78 and/or VL of the antibody comprises an amino acidsequence represented by SEQ ID NO:84, a humanized antibody in which VHof the antibody comprises an amino acid sequence represented by SEQ IDNO: 80 and/or VL of the antibody comprises an amino acid sequencerepresented by SEQ ID NO:84, and a humanized antibody in which VH of theantibody comprises an amino acid sequence represented by SEQ ID NO:82and/or VL of the antibody comprises an amino acid sequence representedby SEQ ID NO:84;[28] A hybridoma which produces the monoclonal antibody described in anyone of [1] to [27];[29] The hybridoma described in [28], in which the hybridoma is ahybridoma KM4008 (FERM BP-10962) or a hybridoma KM4012 (FERM BP-10963)];[30] A DNA which encodes the antibody or the antibody fragment thereofdescribed in any one of [1] to [29];[31] A recombinant vector which comprises the DNA described in [30];[32] A transformant obtainable by introducing the recombinant vectordescribed in [31] into a host cell;[33] A process for producing the antibody or the antibody fragmentthereof described in any one of [1] to [27], comprising culturing thehybridoma described in [28] or [29] or the transformant described in[32] in a medium to form and accumulate the antibody or the antibodyfragment thereof described in any one of [1] to [27] in the culture andcollecting the antibody or the antibody fragment thereof from theculture;[34] A therapeutic agent for ASCT2 related disease comprising theantibody or the antibody fragment thereof described in any one of [1] to[27] as an active ingredient;[35] The therapeutic agent described in [34], in which the diseaserelating to ASCT2 is cancer;[36] The therapeutic agent described in [35], in which the cancer isblood cancer, esophageal cancer, gastric cancer, colorectal cancer,liver cancer or prostate cancer;[37] A method for immunologically detecting or measuring ASCT2, usingthe antibody or the antibody fragment thereof described in any one of[1] to [27];[38] The method described in [37], in which the method forimmunologically measuring is an immunoprecipitation method;[39] A method for immunologically detecting or measuring a cell whichexpresses ASCT2, using the antibody or the antibody fragment thereofdescribed in any one of [1] to [27];[40] The method described in [39], in which the method forimmunologically detecting is a fluorescent cell staining method;[41] A reagent for immunologically detecting or measuring ASCT2 usingantibody or the antibody fragment thereof described in any one of [1] to[27];[42] A diagnostic agent for ASCT2 related disease using the antibody orthe antibody fragment thereof described in any one of [1] to [27];[43] The diagnostic agent described in [42], in which the diseaserelating to ASCT2 is cancer; and[44] The diagnostic agent described in [43], in which the cancer isblood cancer, esophageal cancer, gastric cancer, colorectal cancer,liver cancer or prostate cancer.

The present invention relates to a monoclonal antibody whichspecifically recognizes a native three-dimensional structure of anextracellular region of ASCT2 and binds to the extracellular region, oran antibody fragment thereof.

The species of ASCT2 of the present invention is not particularlylimited. An example of the species may preferably be a mammal,specifically a human.

The amino acid sequence information of ASCT2 is available from a knowndatabase such as NCBI (http://www.ncbi.nlm.nih.gov/). Examples includehuman ASCT2 (NCBI Accession No. NP_(—)005619) represented by SEQ IDNO:2, mouse ASCT2 (NCBI Accession No. NP_(—)033227) represented by SEQID NO:86, and the like.

ASCT2 of the present invention includes a polypeptide comprising theamino acid sequence represented by SEQ ID NO:2 or an amino acid sequencewherein one or more amino acid residue(s) is/are deleted, substitutedand/or added in the amino acid sequence represented by SEQ ID NO:2 andhaving a function of ASCT2. ASCT2 of the present invention also includesa polypeptide having 60% or more homology, preferably 80% or morehomology, more preferably 90% or more homology, further preferably 95%or more homology to the amino acid sequence represented by SEQ ID NO:2and having a function of ASCT2.

In the present invention, the polypeptide comprising an amino acidsequence wherein one or more amino acid residue(s) is/are deleted,substituted and/or added in the amino acid sequence represented by SEQID NO:2 can be obtained by site-specific mutagenesis described inMolecular Cloning, A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press (1989), Current Protocols in Molecular Biology,John Wiley & Sons (1987-1997), Nucleic Acids Research, 10, 6487 (1982),Proc. Natl. Acad. Sci., USA, 79, 6409 (1982), Gene, 34, 315 (1985),Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82,488 (1985), etc. For example, it can be obtained by introducing asite-specific mutation into DNA having the nucleotide sequence encodinga polypeptide having the amino acid sequence represented by SEQ ID NO:2.The number of amino acid residues which are deleted, substituted and/oradded is not specifically limited. The suitable number is 1 to dozens,preferably 1 to 20, more preferably 1 to several, further preferably 1to 5.

Examples of the gene encoding ASCT2 include the nucleotide sequencerepresented by SEQ ID NO:1. Examples of the gene encoding ASCT2 alsoinclude a gene containing a DNA comprising a nucleotide sequence havingdeletion, substitution or addition of one or more nucleotides in thenucleotide sequence represented by SEQ ID NO:1 and encoding apolypeptide having the function of ASCT2; a gene containing a DNAcomprising a nucleotide sequence having at least 60% or higher homology,preferably 80% or higher homology, and more preferably 95% or higherhomology with the nucleotide sequence represented by SEQ ID NO:1 andencoding a polypeptide having the function of ASCT2; a gene comprising aDNA which hybridizes with a DNA having the nucleotide sequencerepresented by SEQ ID NO:1 under stringent conditions and containing aDNA that encodes a polypeptide having the function of ASCT2, and thelike.

In the present invention, the DNA which hybridizes under stringentconditions refers to a DNA which is obtained by colony hybridization,plaque hybridization, Southern hybridization, DNA microarray, or thelike using, a DNA having the nucleotide sequence represented by SEQ IDNO:1 as a probe. A specific example of such DNA is a hybridized colony-or plaque derived DNA which can be identified by performinghybridization at 65° C. in the presence of 0.7 to 1.0 mol/l sodiumchloride using a filter or slide glass with the PCR product or oligo DNAhaving immobilized thereon, and then washing the filter or slide glassat 65° C. with a 0.1 to 2-fold concentration SSC solution (1-foldconcentration SSC solution: 150 mmol/l sodium chloride and 15 mmol/lsodium citrate). Hybridization can be carried out according to themethods described in Molecular Cloning, A Laboratory Manual, SecondEdition, Cold Spring Harbor Lab. Press (1989), Current Protocols inMolecular Biology, John Wiley & Sons (1987-1997); DNA Cloning 1: CoreTechniques, A Practical Approach, Second Edition, Oxford University(1995). Specifically, the DNA capable of hybridization includes DNAhaving at least 60% or more homology, preferably 80% or more homology,more preferably 90% or more homology, and most preferably 95% or morehomology to the nucleotide sequence represented by SEQ ID NO:1.

In the nucleotide sequence of the gene encoding a protein of aeukaryote, genetic polymorphism is often recognized. The ASCT2 gene usedin the present invention also includes a gene in which smallmodifications are generated in the nucleotide sequence by suchpolymorphism.

The number of the homology described in the present invention may be anumber calculated by using a homology search program known by theskilled person, unless otherwise indicated. Regarding the nucleotidesequence, the number may be calculated by BLAST [J. Mol. Biol., 215, 403(1990)] with a default parameter or the like, and regarding the aminoacid sequence, the number may be calculated by using BLAST2 [NucleicAcids Res., 25, 3389 (1997); Genome Res., 7, 649 (1997);http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/information3.html] witha default parameter or the like.

As the default parameter, G (cost to open gap) is 5 for the nucleotidesequence and 11 for the amino acid sequence; -E (cost to extend gap) is2 for the nucleotide sequence and 1 for the amino acid sequence; -q(penalty for nucleotide mismatch) is −3; -r (reward for nucleotidematch) is 1; -e (expect value) is 10; —W (wordsize) is 11 residues forthe nucleotide sequence and 3 residues for the amino acid sequence; -y(dropoff (X) for blast extensions in bits) is 20 for blastn and 7 for aprogram other than blastn; -X (X dropoff value for gapped alignment inbits) is 15; and -Z (final X dropoff value for gapped alignment in bits)is 50 for blastn and 25 for a program other than blastn(http://www.ncbi.nlm.nih.gov/blast/html/blastcgihelp.html).

A polypeptide comprising a partial sequence of the amino acid sequencerepresented by SEQ ID NO:2 can be prepared by methods known to thoseskilled in the art, for example, by partially deleting a DNA encodingthe amino acid sequence represented by SEQ ID NO:2, and culturing atransformant into which an expression vector containing the partiallydeleted DNA is introduced. Based on the thus constructed polypeptide orDNA, a polypeptide in which one or more amino acids are deleted,substituted and/or added in a partial sequence of the amino acidsequence represented by SEQ ID NO:2 can also be obtained, in accordancewith the same method as described above. Further, the polypeptidecomprising a partial sequence of the amino acid sequence represented bySEQ ID NO:2, or the polypeptide in which one or more amino acids aredeleted, substituted and/or added in a partial sequence of the aminoacid sequence represented by SEQ ID NO:2 can also be produced by achemical synthesis method such as a fluorenylmethyloxycarbonyl (Fmoc)method, a t-butyloxycarbonyl (tBoc) method, or the like.

Examples of the extracellular region of ASCT2 in the present inventioninclude a region predicted by a known transmembrane region predictionprograms SOSUI (http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html),TMHMM ver. 2 (http://www.cbs.dtu.dk/services/TMHMM-2.0/), ExPAsyProteomics Server (http://Ca.expasy.org/), or the like, based on theamino acid sequence of the polypeptide represented by SEQ ID NO:2.Specifically, examples include five regions, at positions 74 to 98, atpositions 154 to 224, at positions 287 to 305, at positions 357 to 376and at positions 420 to 425 which are the extracellular region predictedby ExPASy Proteomics Server. Alternatively, examples include fiveregions, at positions 65 to 88, at positions 152 to 224, at positions288 to 306, at positions 361 to 380 and at positions 447 to 451 whichare the extracellular regions predicted in the literature [J. Biol.Chemistry, 271, 18657 (1996)] or [J. Virology, 73, 4470 (1999)]. In thepresent invention, the five extracellular regions are indicated as anEL1 region, an EL2 region, an EL3 region, an EL4 region and an EL5region sequentially from the N-terminal side. For example, the EL2region in an amino acid sequence of the ASCT2 polypeptide represented bySEQ ID NO:2 corresponds to positions 154 to 224 or positions 152 to 224.

The monoclonal antibody of the present invention binds to anextracellular region of ASCT2, preferably any one of the above-mentionedEL1 to EL5 regions, and more preferably the EL2 region.

The native three-dimensional structure of the extracellular region ofASCT2 in the present invention may be any structure, so long as it has athree-dimensional structure equivalent to a three-dimensional structurewhich can be formed in a native state by the extracellular region ofASCT2 having the amino acid sequence represented by SEQ ID NO:2.

Whether the antibody or the antibody fragment thereof of the presentinvention specifically recognizes a native three-dimensional structureof an extracellular region of ASCT2 and binds to the extracellularregion can be confirmed by radioimmunoassay using a solid-phase sandwichmethod or the like, or by a known immunological detection method forASCT2-expressing cells using such as enzyme immunoassay (ELISA) methodor the like, preferably a method capable of investigating a binding ofan antibody to a particular antigen and a cell expressing the particularantigen, such as a fluorescent cell staining method. For example,specific examples include a fluorescent antibody staining method usingan FMAT8100HTS System (manufactured by Applied Biosystems) [CancerImmunol. Immunother., 36, 373 (1993)] or the like, a fluorescent cellstaining method using flow cytometry, surface plasmon resonance using aBiacore System (manufactured by GE Healthcare) or the like, or othermethods. Furthermore, a known immunological detection methods[Monoclonal Antibodies—Principles and practice, Third edition, AcademicPress (1996), Antibodies—A Laboratory Manual, Cold Spring HarborLaboratory (1988), Monoclonal Antibody Experimental Manual, Kodan-shaScientific (1987)] and the like can be combined to confirm the binding.

In the present invention, the cell expressing ASCT2 may be any cell, solong as it expresses ASCT2, and examples include a cell which isnaturally present in the human body, a cell line established from thecell which is naturally present in the human body, a cell obtained bygene recombination technique and the like.

The cell which is naturally present in the human body includes a cellexpressing the polypeptide in the body of a cancer patient, such as acell expressing the ASCT2 among tumor cells obtained by biopsy or thelike.

Example of a cell line established from the cell which is naturallypresent in the human body include a cell line expressing ASCT2, amongcell lines prepared by establishing the ASCT2-expressing cells obtainedfrom the above cancer patients, and examples include the multiplemyeloma cell line KMS-11 [Human Science Research Resources Bank (HSRRB)Accession No. JCRB1179], RPMI 8226 (HSRRB Accession No. JCRB0034) andthe like.

Specific examples of the cell obtained by gene recombination techniquesmay include an ASCT2-expressing cell obtained by introducing anexpression vector comprising an ASCT2-encoding cDNA into an insect cell,an animal cell or the like, and others.

The monoclonal antibody of the present invention includes an antibodyproduced by a hybridoma and a recombinant antibody produced by atransformant transformed with an expression vector containing a geneencoding an antibody.

The hybridoma can be prepared, for example, by preparing the above cellexpressing ASCT2 as an antigen, inducing an antibody-producing cellhaving antigen specificity from an animal immunized with the antigen,and fusing the antigen-producing cell with a myeloma cell. Theanti-ASCT2 antibody can be obtained by culturing the hybridoma oradministering the hybridoma cell into an animal to cause ascites tumorin the animal and separating and purifying the culture or the ascites.

The animal immunized with an antigen may be any animal, so long as ahybridoma can be prepared, and mouse, rat, hamster, rabbit or the likeis suitably used. Also, the antibody of the present invention includesan antibody produced by a hybridoma obtained by fusion of the cellhaving antibody-producing activity can be obtained from such an animaland immune in vitro with a myeloma cell.

Specific examples of the monoclonal antibody produced by the hybridomaof the present invention may include an antibody KM3998 produced by ahybridoma KM3998, an antibody KM4000 produced by a hybridoma KM4000, anantibody

KM4001 produced by a hybridoma KM4001, an antibody KM4008 produced by ahybridoma KM4008, an antibody KM4012 produced by a hybridoma KM4012, anantibody KM4018 produced by a hybridoma KM4018, and the like. Thehybridomas KM4008 and KM4012 have been deposited to National Instituteof Advanced Industrial Science and Technology under the Budapest Treaty,on May 1, 2008, as FERM BP-10962 and FERM BP-10963, respectively.

Further, the monoclonal antibody of the present invention also includesa monoclonal antibody that binds to an epitope to which an antibodyproduced from the above-mentioned hybridomas binds.

The recombinant antibody of the present invention includes an antibodyproduced by gene recombination, such as a human chimeric antibody, ahumanized antibody, a human antibody and an antibody fragment thereof.Among the recombinant antibodies, one having character of a commonmonoclonal antibody, low immunogenecity and prolonged half-life in bloodis preferable as a therapeutic agent.

Examples of the recombinant antibody include a recombinant antibodymodified the above mentioned monoclonal antibody by using recombinanttechnologies.

Examples of the recombinant antibody of the present invention include arecombinant antibody in which Complementarity Determining Region(hereinafter referred to as CDR) 1, CDR2 and CDR3 of a heavy chainvariable region (hereinafter, referred to as VH) of the antibodycomprise the amino acid sequences represented by SEQ ID NOs:26, 27 and28, respectively, and/or CDR1, CDR2 and CDR3 of a light chain variableregion (hereinafter, referred to as VL) of the antibody comprise theamino acid sequences represented by SEQ ID NOs:29, 30 and 31,respectively; a recombinant antibody in which CDR1, CDR2 and CDR3 of VHof the antibody comprise the amino acid sequences represented by SEQ IDNOs:32, 33 and 34, respectively, and/or CDR1, CDR2 and CDR3 of VL of theantibody comprise the amino acid sequences represented by SEQ ID NOs:35,36 and 37, respectively; a recombinant antibody in which CDR1, CDR2 andCDR3 of VH of the antibody comprise the amino acid sequences representedby SEQ ID NOs:49, 50 and 51, respectively, and/or CDR1, CDR2 and CDR3 ofVL of the antibody comprise the amino acid sequences represented by SEQID NOs:52, 53 and 54, respectively; and the like.

The human chimeric antibody is an antibody comprising VH and VL of anantibody of a non-human animal, and a heavy chain constant region(hereinafter referred to as CH) and a light chain constant region(hereinafter referred to as CL) of a human antibody. Specifically, thehuman chimeric antibody of the present invention can be produced byobtaining cDNAs encoding VH and VL from a hybridoma which produces amonoclonal antibody which specifically recognizes a nativethree-dimensional structure of an extracellular region of ASCT2 andbinds to the extracellular region, inserting each of them into anexpression vector for animal cell comprising DNAs encoding CH and CL ofhuman antibody to thereby construct a vector for expression of humanchimeric antibody, and then introducing the vector into an animal cellto express the antibody.

As the CH of the human chimeric antibody, any CH can be used, so long asit belongs to human immunoglobulin (hereinafter referred to as “hIg”),and those belonging to the hIgG class are preferred, and any one of thesubclasses belonging to the hIgG class, such as hIgG1, hIgG2, hIgG3 andhIgG4, can be used. As the CL of the human chimeric antibody, any CL canbe used, so long as it belongs to the hIg class, and those belonging toκ class or γ class can be used.

Examples of the human chimeric antibody of the present invention includea human chimeric antibody in which VH of the antibody comprises theamino acid sequences represented by SEQ ID NO:19, and VL of the antibodycomprises the amino acid sequences represented by SEQ ID NO:21; a humanchimeric antibody in which VII of the antibody comprises the amino acidsequence represented by SEQ ID NO:23, and VL of the antibody comprisesthe amino acid sequence represented by SEQ ID NO:25; a human chimericantibody in which VH of the antibody comprises the amino acid sequencerepresented by SEQ ID NO:46, and VL of the antibody comprises the aminoacid sequence represented by SEQ ID NO:48; and the like. Specificexamples include human chimeric antibodies cKM4008, cKM4012, cKM4018 andthe like.

A humanized antibody is an antibody in which amino acid sequences ofCDRs of VH and VL of an antibody derived from a non-human animal aregrafted into appropriate positions of VH and VL of a human antibody, andis also called a human CDR-grafted antibody, a reshaped-antibody or thelike. The humanized antibody of the present invention can be produced byconstructing cDNAs encoding an antibody variable region (hereinafterreferred to as “V region”) in which the amino acid sequences of CDRs ofVH and VL of an antibody derived from a non-human animal produced by ahybridoma which produces a monoclonal antibody which specificallyrecognizes three-dimensional structure of ASCT2 and binds to theextracellular region are grafted into frameworks (hereinafter referredto as “FR”) of VH and VL of a suitable human antibody, inserting each ofthem into a vector for expression of animal cell comprising genesencoding CH and CL of a human antibody to thereby construct a vector forexpression of humanized antibody, and introducing it into an animal cellto thereby express and produce the humanized antibody.

As the amino acid sequences of FRs of VH and VL of a humanized antibody,any amino acid sequences can be used, so long as they are amino acidsequences of FRs of VH and VL, respectively, derived from a humanantibody. Examples include amino acid sequences of FRs of VH and VL ofhuman antibodies registered in database such as Protein Data Bank,common amino acid sequences of each sub group of FRs of VH and VL ofhuman antibodies described in, for example, Sequences of Proteins ofImmunological Interest, US Dept. Health and Human Services (1991), andthe like.

As the CH of the humanized antibody, any CH can be used, so long as itbelongs to the Mg class, and those of the hIgG class are preferred andany one of the subclasses belonging to the hIgG class, such as hIgG1,hIgG2, hIgG3 and hIgG4 can be used. As the CL of the humanized antibody,any CL can be used, so long as it belongs to the Mg class, and thosebelonging to the κ class or γ class can be used.

Specific examples of the humanized antibody of the present inventioninclude a humanized antibody wherein VH of the antibody comprises theamino acid sequence represented by SEQ ID NO:71, or an amino acidsequence in which at least one amino acid residue selected from Val atposition 2, Ser at position 9, Val at position 20, Ser at position 30,Arg at position 38, Glu at position 46, Leu at position 86, Val atposition 93, Tyr at position 95 and Val at position 116 in the aminoacid sequence represented by SEQ ID NO:71 is substituted with otheramino acid residue(s), and/or wherein VL of the antibody comprises theamino acid sequence represented by SEQ ID NO:72, or an amino acidsequence in which at least one amino acid residue selected from Pro atposition 8, Val at position 15, Gln at position 38, Ala at position 43,Pro at position 44, Phe at position 71 and Tyr at position 87 in theamino acid sequence represented by SEQ ID NO:72 is substituted withother amino acid residue(s); and the like. In this connection, thenumber of modifications which are introduced is not limited.

Examples of the amino acid sequence of VH of the antibody includepreferably:

a humanized antibody comprising an amino acid sequence in which Ser atposition 9, Val at position 20, Arg at position 38, Glu at position 46,Val at position 93, Tyr at position 95 and Val at position 116 in theamino acid sequence represented by SEQ ID NO:71 are substituted withother amino acid residues,

a humanized antibody comprising an amino acid sequence in which Val atposition 20, Glu at position 46, Tyr at position 95 and Val at position116 in amino acid sequence represented by SEQ ID NO:71 are substitutedwith other amino acid residues, or

a humanized antibody comprising an amino acid sequence in which Glu atposition 46, and Tyr at position 95 in the amino acid sequencerepresented by SEQ ID NO:71 are substituted with other amino acidresidues.

The amino acid sequence of VH of the antibody obtained by the aboveamino acid modifications may include, for example, an amino acidsequence in which at least one modification selected from amino acidmodifications for substituting Val at position 2 with Ile, Ser atposition 9 with Pro, Val at position 20 with Ile, Ser at position 30with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leuat position 86 with Val, Val at position 93 with Thr, Tyr at position 95with Phe, and Val at position 116 with Leu is introduced in the aminoacid sequence represented by SEQ ID NO:71.

Specific example of the amino acid sequence of VH in which tenmodifications are introduced include an amino acid sequence in whichsubstitutions of Val at position 2 with Ile, Ser at position 9 with Pro,Val at position 20 with Ile, Ser at position 30 with Thr, Arg atposition 38 with Lys, Glu at position 46 with Lys, Leu at position 86with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, andVal at position 116 with Leu are introduced in the amino acid sequencerepresented by SEQ ID NO:71.

Specific examples of the amino acid sequence of VH in which ninemodifications are introduced in the amino acid sequence represented bySEQ ID NO:71 include the following amino acid sequences:

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg atposition 38 with Lys, Glu at position 46 with Lys, Leu at position 86with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, andVal at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Val at position 20 with Ile, Ser at position 30 with Thr, Arg atposition 38 with Lys, Glu at position 46 with Lys, Leu at position 86with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, andVal at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Ser at position 30 with Thr, Arg atposition 38 with Lys, Glu at position 46 with Lys, Leu at position 86with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, andVal at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Arg atposition 38 with Lys, Glu at position 46 with Lys, Leu at position 86with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, andVal at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Ser atposition 30 with Thr, Glu at position 46 with Lys, Leu at position 86with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, andVal at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Ser atposition 30 with Thr, Arg at position 38 with Lys, Leu at position 86with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, andVal at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Ser atposition 30 with Thr, Arg at position 38 with Lys, Glu at position 46with Lys, Val at position 93 with Thr, Tyr at position 95 with Phe, andVal at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Ser atposition 30 with Thr, Arg at position 38 with Lys, Glu at position 46with Lys, Leu at position 86 with Val, Tyr at position 95 with Phe, andVal at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Ser atposition 30 with Thr, Arg at position 38 with Lys, Glu at position 46with Lys, Leu at position 86 with Val, Val at position 93 with Thr, andVal at position 116 with Leu are introduced, and

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Ser atposition 30 with Thr, Arg at position 38 with Lys, Glu at position 46with Lys, Leu at position 86 with Val, Val at position 93 with Thr, andTyr at position 95 with Phe are introduced.

Specific examples of the amino acid sequence of VH in which eightmodifications are introduced in the amino acid sequence represented bySEQ ID NO:71 include the following amino acid sequences:

an amino acid sequence in which substitutions of Val at position 20 withIle, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu atposition 46 with Lys, Leu at position 86 with Val, Val at position 93with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu atposition 46 with Lys, Leu at position 86 with Val, Val at position 93with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 20 with Ile, Arg at position 38 with Lys, Glu atposition 46 with Lys, Leu at position 86 with Val, Val at position 93with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 20 with Ile, Ser at position 30 with Thr, Glu atposition 46 with Lys, Leu at position 86 with Val, Val at position 93with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg atposition 38 with Lys, Leu at position 86 with Val, Val at position 93with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg atposition 38 with Lys, Glu at position 46 with Lys, Val at position 93with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg atposition 38 with Lys, Glu at position 46 with Lys, Leu at position 86with Val, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg atposition 38 with Lys, Glu at position 46 with Lys, Leu at position 86with Val, Val at position 93 with Thr, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg atposition 38 with Lys, Glu at position 46 with Lys, Leu at position 86with Val, Val at position 93 with Thr, and Tyr at position 95 with Pheare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu atposition 46 with Lys, Leu at position 86 with Val, Val at position 93with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Val at position 20 with Ile, Arg at position 38 with Lys, Glu atposition 46 with Lys, Leu at position 86 with Val, Val at position 93with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Val at position 20 with Ile, Ser at position 30 with Thr, Glu atposition 46 with Lys, Leu at position 86 with Val, Val at position 93with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Val at position 20 with Ile, Ser at position 30 with Thr, Arg atposition 38 with Lys, Leu at position 86 with Val, Val at position 93with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Val at position 20 with Ile, Ser at position 30 with Thr, Arg atposition 38 with Lys, Glu at position 46 with Lys, Val at position 93with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Val at position 20 with Ile, Ser at position 30 with Thr, Arg atposition 38 with Lys, Glu at position 46 with Lys, Leu at position 86with Val, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Val at position 20 with Ile, Ser at position 30 with Thr, Arg atposition 38 with Lys, Glu at position 46 with Lys, Leu at position 86with Val, Val at position 93 with Thr, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Val at position 20 Ile, Ser at position 30 with Thr, Arg atposition 38 with Lys, Glu at position 46 with Lys, Leu at position 86with Val, Val at position 93 with Thr, and Tyr at position 95 with Pheare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Arg at position 38 with Lys, Glu atposition 46 with Lys, Leu at position 86 with Val, Val at position 93with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Ser at position 30 with Thr, Glu atposition 46 with Lys, Leu at position 86 with Val, Val at position 93with Thr, Tyr at position 95 with Phe and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Ser at position 30 with Thr, Arg atposition 38 with Lys, Leu at position 86 with Val, Val at position 93with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Ser at position 30 with Thr, Arg atposition 38 with Lys, Glu at position 46 with Lys, Val at position 93with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Ser at position 30 with Thr, Arg atposition 38 with Lys, Glu at position 46 with Lys, Leu at position 86with Val, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Ser at position 30 with Thr, Arg atposition 38 with Lys, Glu at position 46 with Lys, Leu at position 86with Val, Val at position 93 with Thr, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Ser at position 30 with Thr, Arg atposition 38 with Lys, Glu at position 46 with Lys, Leu at position 86with Val, Val at position 93 with Thr, and Tyr at position 95 with Pheare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Glu atposition 46 with Lys, Leu at position 86 with Val, Val at position 93with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Arg atposition 38 with Lys, Leu at position 86 with Val, Val at position 93with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Arg atposition 38 with Lys, Glu at position 46 with Lys, Val at position 93with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Arg atposition 38 with Lys, Glu at position 46 with Lys, Leu at position 86with Val, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Arg atposition 38 with Lys, Glu at position 46 with Lys, Leu at position 86with Val, Val at position 93 with Thr, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Arg atposition 38 with Lys, Glu at position 46 with Lys, Leu at position 86with Val, Val at position 93 with Thr, and Tyr at position 95 with Pheare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Ser atposition 30 with Thr, Leu at position 86 with Val, Val at position 93with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Ser atposition 30 with Thr, Glu at position 46 with Lys, Val at position 93with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Ser atposition 30 with Thr, Glu at position 46 with Lys, Leu at position 86with Val, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Ser atposition 30 with Thr, Glu at position 46 with Lys, Leu at position 86with Val, Val at position 93 with Thr, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Ser atposition 30 with Thr, Glu at position 46 with Lys, Leu at position 86with Val, Val at position 93 with Thr, and Tyr at position 95 with Pheare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Ser atposition 30 with Thr, Arg at position 38 with Lys, Val at position 93with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Ser atposition 30 with Thr, Arg at position 38 with Lys, Leu at position 86with Val, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Ser atposition 30 with Thr, Arg at position 38 with Lys, Leu at position 86with Val, Val at position 93 with Thr, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Ser atposition 30 with Thr, Arg at position 38 with Lys, Leu at position 86with Val, Val at position 93 with Thr, and Tyr at position 95 with Pheare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Ser atposition 30 with Thr, Arg at position 38 with Lys, Glu at position 46with Lys, Tyr at position 95 with Phe, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Ser atposition 30 with Thr, Arg at position 38 with Lys, Glu at position 46with Lys, Val at position 93 with Thr, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 Ile, Ser at position30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys,Val at position 93 with Thr, and Tyr at position 95 with Phe areintroduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Ser atposition 30 with Thr, Arg at position 38 with Lys, Glu at position 46with Lys, Leu at position 86 with Val, and Val at position 116 with Leuare introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Ser atposition 30 with Thr, Arg at position 38 with Lys, Glu at position 46with Lys, Leu at position 86 with Val, and Tyr at position 95 with Pheare introduced, and

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Ser atposition 30 with Thr, Arg at position 38 with Lys, Glu at position 46with Lys, Leu at position 86 with Val, and Val at position 93 with Thrare introduced.

Specific examples of the amino acid sequence of VH in which sevenmodifications are introduced in the amino acid sequence represented bySEQ ID NO:71 include the following amino acid sequences:

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Ser atposition 30 with Thr, Glu at position 46 with Lys, Tyr at position 95with Phe, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Arg atposition 38 with Lys, Glu at position 46 with Lys, Tyr at position 95with Phe, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Glu atposition 46 with Lys, Leu at position 86 with Val, Tyr at position 95with Phe, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Ser at position 9 with Pro, Val at position 20 with Ile, Glu atposition 46 with Lys, Val at position 93 with Thr, Tyr at position 95with Phe, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Val at position 20 with Ile, Ser at position 30 with Thr, Arg atposition 38 with Lys, Glu at position 46 with Lys, Tyr at position 95with Phe, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Val at position 20 with Ile, Ser at position 30 with Thr, Glu atposition 46 with Lys, Leu at position 86 with Val, Tyr at position 95with Phe, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Val at position 20 with Ile, Ser at position 30 with Thr, Glu atposition 46 with Lys, Val at position 93 with Thr, Tyr at position 95with Phe, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Val at position 20 with Ile, Arg at position 38 with Lys, Glu atposition 46 with Lys, Leu at position 86 with Val, Tyr at position 95with Phe, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Val at position 20 with Ile, Arg at position 38 with Lys, Glu atposition 46 with Lys, Val at position 93 with Thr, Tyr at position 95with Phe, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, Val at position 20 with Ile, Glu at position 46 with Lys, Leu atposition 86 with Val, Val at position 93 with Thr, Tyr at position 95with Phe, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg atposition 38 with Lys, Glu at position 46 with Lys, Tyr at position 95with Phe, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 20 with Ile, Ser at position 30 with Thr, Glu atposition 46 with Lys, Leu at position 86 with Val, Tyr at position 95with Phe, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 20 with Ile, Ser at position 30 with Thr, Glu atposition 46 with Lys, Val at position 93 with Thr, Tyr at position 95with Phe, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 20 with Ile, Arg at position 38 with Lys, Glu atposition 46 with Lys, Leu at position 86 with Val, Tyr at position 95with Phe, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 20 with Ile, Arg at position 38 with Lys, Glu atposition 46 with Lys, Val at position 93 with Thr, Tyr at position 95with Phe, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu atposition 46 with Lys, Leu at position 86 with Val, Tyr at position 95with Phe, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu atposition 46 with Lys, Val at position 93 with Thr, Tyr at position 95with Phe, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, Ser at position 30 with Thr, Glu at position 46 with Lys, Leu atposition 86 with Val, Val at position 93 with Thr, Tyr at position 95with Phe, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu atposition 86 with Val, Val at position 93 with Thr, Tyr at position 95with Phe, and Val at position 116 with Leu are introduced, and the like.

Specific examples of the amino acid sequence of VH in which sixmodifications are introduced in the amino acid sequence represented bySEQ ID NO:71 include the following amino acid sequences:

an amino acid sequence in which substitutions of Val at position 20 withIle, Arg at position 38 with Lys, Glu at position 46 with Lys, Val atposition 93 with Thr, Tyr at position 95 with Phe, and Val at position116 with Leu are introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Arg at position 38 with Lys, Glu at position 46 with Lys, Val atposition 93 with Thr, Tyr at position 95 with Phe, and Val at position116 with Leu are introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 20 with Ile, Glu at position 46 with Lys, Val atposition 93 with Thr, Tyr at position 95 with Phe, and Val at position116 with Leu are introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 20 with Ile, Arg at position 38 with Lys, Val atposition 93 with Thr, Tyr at position 95 with Phe, and Val at position116 with Leu are introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 20 with Ile, Arg at position 38 with Lys, Glu atposition 46 with Lys, Tyr at position 95 with Phe, and Val at position116 with Leu are introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 20 with Ile, Arg at position 38 with Lys, Glu atposition 46 with Lys, Val at position 93 with Thr, and Val at position116 with Leu are introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 20 with Ile, Arg at position 38 with Lys, Glu atposition 46 with Lys, Val at position 93 with Thr, and Tyr at position95 with Phe are introduced, and the like.

Specific examples of the amino acid sequence of VH in which fivemodifications are introduced in the amino acid sequence represented bySEQ ID NO:71 include the following amino acid sequences:

an amino acid sequence in which substitutions of Val at position 2 withIle, Val at position 20 with Ile, Glu at position 46 with Lys, Tyr atposition 95 with Phe, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 20 with Ile, Glu at position 46 with Lys, Tyr atposition 95 with Phe, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, Ser at position 30 with Thr, Glu at position 46 with Lys, Tyr atposition 95 with Phe, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, Arg at position 38 with Lys, Glu at position 46 with Lys, Tyr atposition 95 with Phe, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, Glu at position 46 with Lys, Leu at position 86 with Val, Tyr atposition 95 with Phe, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, Glu at position 46 with Lys, Tyr at position 95 with Phe, and Valat position 116 with Leu are introduced, and the like.

Specific examples of the amino acid sequence of VH in which fourmodifications are introduced in the amino acid sequence represented bySEQ ID NO:71 include the following amino acid sequences:

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 20 with Ile, Glu at position 46 with Lys, and Tyrat position 95 with Phe are introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Arg at position 38 with Lys, Glu at position 46 with Lys, and Tyrat position 95 with Phe are introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Glu at position 46 with Lys, Val at position 93 with Thr, and Tyrat position 95 with Phe are introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Glu at position 46 with Lys, Tyr at position 95 with Phe, and Valat position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, Arg at position 38 with Lys, Glu at position 46 with Lys, and Tyrat position 95 with Phe are introduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, Glu at position 46 with Lys, Val at position 93 with Thr, and Tyrat position 95 with Phe are introduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, Glu at position 46 with Lys, Tyr at position 95 with Phe, and Valat position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Arg at position 38 withLys, Glu at position 46 with Lys, Val at position 93 with Thr, and Tyrat position 95 with Phe are introduced,

an amino acid sequence in which substitutions of Arg at position 38 withLys, Glu at position 46 with Lys, Tyr at position 95 with Phe, and Valat position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Glu at position 46 withLys, Val at position 93 with Thr, Tyr at position 95 with Phe, and Valat position 116 with Leu are introduced, and the like.

Specific examples of the amino acid sequence of VH in which threemodifications are introduced in the amino acid sequence represented bySEQ ID NO:71 include the following amino acid sequences:

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 20 with Ile, and Arg at position 38 with Lys areintroduced, an amino acid sequence in which substitutions of Ser atposition 9 with Pro, Val at position 20 with Ile, and Glu at position 46with Lys are introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 20 with Ile, and Val at position 93 with Thr areintroduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 20 with Ile, and Tyr at position 95 with Phe areintroduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 20 with Ile, and Val at position 116 with Leu areintroduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Arg at position 38 with Lys, and Glu at position 46 with Lys areintroduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Arg at position 38 with Lys, and Val at position 93 with Thr areintroduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Arg at position 38 with Lys, and Tyr at position 95 with Phe areintroduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Arg at position 38 with Lys, and Val at position 116 with Leu areintroduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Glu at position 46 with Lys, and Val at position 93 with Thr areintroduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Glu at position 46 with Lys, and Tyr at position 95 with Phe areintroduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Glu at position 46 with Lys, and Val at position 116 with Leu areintroduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 93 with Thr, and Tyr at position 95 with Phe areintroduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Val at position 93 with Thr, and Val at position 116 with Leu areintroduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, Tyr at position 95 with Phe, and Val at position 116 with Leu areintroduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, Arg at position 38 with Lys, and Glu at position 46 with Lys areintroduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, Arg at position 38 with Lys, and Val at position 93 with Thr areintroduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, Arg at position 38 with Lys, and Tyr at position 95 with Phe areintroduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, Arg at position 38 with Lys, and Val at position 116 with Leu areintroduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, Glu at position 46 with Lys, and Val at position 93 with Thr areintroduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, Glu at position 46 with Lys, and Tyr at position 95 with Phe areintroduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, Glu at position 46 with Lys, and Val at position 116 with Leu areintroduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, Val at position 93 with Thr, and Tyr at position 95 with Phe areintroduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, Val at position 93 with Thr, and Val at position 116 with Leu areintroduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, Tyr at position 95 with Phe, and Val at position 116 with Leu areintroduced,

an amino acid sequence in which substitutions of Arg at position 38 withLys, Glu at position 46 with Lys, and Val at position 93 with Thr areintroduced,

an amino acid sequence in which substitutions of Arg at position 38 withLys, Glu at position 46 with Lys, and Tyr at position 95 with Phe areintroduced,

an amino acid sequence in which substitutions of Arg at position 38 withLys, Glu at position 46 with Lys, and Val at position 116 with Leu areintroduced,

an amino acid sequence in which substitutions of Arg at position 38 withLys, Val at position 93 with Thr, and Tyr at position 95 with Phe areintroduced,

an amino acid sequence in which substitutions of Arg at position 38 withLys, Val at position 93 with Thr, and Val at position 116 with Leu areintroduced,

an amino acid sequence in which substitutions of Arg at position 38 withLys, Tyr at position 95 with Phe, and Val at position 116 with Leu areintroduced,

an amino acid sequence in which substitutions of Glu at position 46 withLys, Val at position 93 with Thr, and Tyr at position 95 with Phe areintroduced,

an amino acid sequence in which substitutions of Glu at position 46 withLys, Val at position 93 with Thr, and Val at position 116 with Leu areintroduced,

an amino acid sequence in which substitutions of Glu at position 46 withLys, Tyr at position 95 with Phe, and Val at position 116 with Leu areintroduced,

an amino acid sequence in which substitutions of Val at position 93 withThr, Tyr at position 95 with Phe, and Val at position 116 with Leu areintroduced, and the like.

Specific examples of the amino acid sequence of VH in which twomodifications are introduced in the amino acid sequence represented bySEQ ID NO:71 include the following amino acid sequences:

an amino acid sequence in which substitutions of Val at position 2 withIle, and Ser at position 9 with Pro are introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, and Val at position 20 with Ile are introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, and Ser at position 30 with Thr are introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, and Arg at position 38 with Lys are introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, and Glu at position 46 with Lys are introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, and Leu at position 86 with Val are introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, and Val at position 93 with Thr are introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, and Tyr at position 95 with Phe are introduced,

an amino acid sequence in which substitutions of Val at position 2 withIle, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, and Val at position 20 with Ile are introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, and Ser at position 30 with Thr are introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, and Arg at position 38 with Lys are introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, and Glu at position 46 with Lys are introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, and Leu at position 86 with Val are introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, and Val at position 93 with Thr are introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, and Tyr at position 95 with Phe are introduced,

an amino acid sequence in which substitutions of Ser at position 9 withPro, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, and Ser at position 30 with Thr are introduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, and Arg at position 38 with Lys are introduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, and Glu at position 46 with Lys are introduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, and Leu at position 86 with Val are introduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, and Val at position 93 with Thr are introduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, and Tyr at position 95 with Phe are introduced,

an amino acid sequence in which substitutions of Val at position 20 withIle, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Ser at position 30 withThr, and Arg at position 38 with Lys are introduced,

an amino acid sequence in which substitutions of Ser at position 30 withThr, and Glu at position 46 with Lys are introduced,

an amino acid sequence in which substitutions of Ser at position 30 withThr, and Leu at position 86 with Val are introduced,

an amino acid sequence in which substitutions of Ser at position 30 withThr, and Val at position 93 with Thr are introduced,

an amino acid sequence in which substitutions of Ser at position 30 withThr, and Tyr at position 95 with Phe are introduced,

an amino acid sequence in which substitutions of Ser at position 30 withThr, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Arg at position 38 withLys, and Glu at position 46 with Lys are introduced,

an amino acid sequence in which substitutions of Arg at position 38 withLys, and Leu at position 86 with Val are introduced,

an amino acid sequence in which substitutions of Arg at position 38 withLys, and Val at position 93 with Thr are introduced,

an amino acid sequence in which substitutions of Arg at position 38 withLys, and Tyr at position 95 with Phe are introduced,

an amino acid sequence in which substitutions of Arg at position 38 withLys, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Glu at position 46 withLys, and Leu at position 86 with Val are introduced,

an amino acid sequence in which substitutions of Glu at position 46 withLys, and Val at position 93 with Thr are introduced,

an amino acid sequence in which substitutions of Glu at position 46 withLys, and Tyr at position 95 with Phe are introduced,

an amino acid sequence in which substitutions of Glu at position 46 withLys, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Leu at position 86 withVal, and Val at position 93 with Thr are introduced,

an amino acid sequence in which substitutions of Leu at position 86 withVal, and Tyr at position 95 with Phe are introduced,

an amino acid sequence in which substitutions of Leu at position 86 withVal, and Val at position 116 with Leu are introduced,

an amino acid sequence in which substitutions of Val at position 93 withThr, and Tyr at position 95 with Phe are introduced,

an amino acid sequence in which substitutions of Val at position 93 withThr, and Val at position 116 with Leu are introduced, and

an amino acid sequence in which substitutions of Tyr at position 95 withPhe, and Val at position 116 with Leu are introduced.

Specific examples of the amino acid sequence of VH in which onemodification is introduced in the amino acid sequence represented by SEQID NO:71 include the following amino acid sequences:

an amino acid sequence in which a substitution of Val at position 2 withIle is introduced,

an amino acid sequence in which a substitution of Ser at position 9 withPro is introduced,

an amino acid sequence in which a substitution of Val at position 20with Ile is introduced,

an amino acid sequence in which a substitution of Ser at position 30with Thr is introduced,

an amino acid sequence in which a substitution of Arg at position 38with Lys is introduced,

an amino acid sequence in which a substitution of Glu at position 46with Lys is introduced,

an amino acid sequence in which a substitution of Leu at position 86with Val is introduced,

an amino acid sequence in which a substitution of Val at position 93with Thr is introduced,

an amino acid sequence in which a substitution of Tyr at position 95with Phe is introduced, and

an amino acid sequence in which a substitution of Val at position 116with Leu is introduced.

With regard to VL of the antibody, preferable examples include

a humanized antibody comprising an amino acid sequence in which Val atposition 15, Ala at position 43, Pro at position 44, Phe at position 71,and Tyr at position 87 in the amino acid sequence represented by SEQ IDNO:72 are substituted with other amino acid residues,

a humanized antibody comprising an amino acid sequence in which Val atposition 15, Phe at position 71 and Tyr at position 87 in amino acidsequence represented by SEQ ID NO:72 are substituted with other aminoacid residues, and the like.

The amino acid sequence of VL of the antibody obtained by the aboveamino acid modifications include, for example, an amino acid sequence inwhich at least one modification selected from amino acid modificationsfor substituting Pro at position 8 with Thr, Val at position 15 withLeu, Gln at position 38 with Arg, Ala at position 43 with Thr, Pro atposition 44 with Val, Phe at position 71 with Tyr, and Tyr at position87 with Phe is introduced in the amino acid sequence represented by SEQID NO:72.

Specific examples of the amino acid sequence of VL in which sevenmodifications are introduced include an amino acid sequence in whichsubstitutions of Pro at position 8 with Thr, Val at position 15 withLeu, Gln at position 38 with Arg, Ala at position 43 with Thr, Pro atposition 44 with Val, Phe at position 71 with Tyr, and Tyr at position87 with Phe are introduced in the amino acid sequence represented by SEQID NO:72.

Specific examples of the amino acid sequence of VL in which sixmodifications are introduced in the amino acid sequence represented bySEQ ID NO:72 include the following amino acid sequences:

an amino acid sequence in which substitutions of Val at position 15 withLeu, Gln at position 38 with Arg, Ala at position 43 with Thr, Pro atposition 44 with Val, Phe at position 71 with Tyr, and Tyr at position87 with Phe are introduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, Gln at position 38 with Arg, Ala at position 43 with Thr, Pro atposition 44 with Val, Phe at position 71 with Tyr, and Tyr at position87 with Phe are introduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, Val at position 15 with Leu, Ala at position 43 with Thr, Pro atposition 44 with Val, Phe at position 71 with Tyr, and Tyr at position87 with Phe are introduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, Val at position 15 with Leu, Gln at position 38 with Arg, Pro atposition 44 with Val, Phe at position 71 with Tyr, and Tyr at position87 with Phe are introduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, Val at position 15 with Leu, Gln at position 38 with Arg, Ala atposition 43 with Thr, Phe at position 71 with Tyr, and Tyr at position87 with Phe are introduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, Val at position 15 with Leu, Gln at position 38 with Arg, Ala atposition 43 with Thr, Pro at position 44 with Val, and Tyr at position87 with Phe are introduced, and

an amino acid sequence in which substitutions of Pro at position 8 withThr, Val at position 15 with Leu, Gln at position 38 with Arg, Ala atposition 43 with Thr, Pro at position 44 with Val, and Phe at position71 with Tyr are introduced.

Specific examples of the amino acid sequence of VL in which fivemodifications are introduced in the amino acid sequence represented bySEQ ID NO:72 include the following amino acid sequences:

an amino acid sequence in which substitutions of Gln at position 38 withArg, Ala at position 43 with Thr, Pro at position 44 with Val, Phe atposition 71 with Tyr, and Tyr at position 87 with Phe are introduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, Ala at position 43 with Thr, Pro at position 44 with Val, Phe atposition 71 with Tyr, and Tyr at position 87 with Phe are introduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, Gln at position 38 with Mg, Pro at position 44 with Val, Phe atposition 71 with Tyr, and Tyr at position 87 with Phe are introduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, Gln at position 38 with Mg, Ala at position 43 with Thr, Phe atposition 71 with Tyr, and Tyr at position 87 with Phe are introduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, Gln at position 38 with Arg, Ala at position 43 with Thr, Pro atposition 44 with Val, and Tyr at position 87 with Phe are introduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, Gin at position 38 with Mg, Ala at position 43 with Thr, Pro atposition 44 with Val, and Phe at position 71 with Tyr are introduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, Ma at position 43 with Thr, Pro at position 44 with Val, Phe atposition 71 with Tyr, and Tyr at position 87 with Phe are introduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, Gln at position 38 with Mg, Pro at position 44 with Val, Phe atposition 71 with Tyr, and Tyr at position 87 with Phe are introduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, Gln at position 38 with Mg, Ala at position 43 with Thr, Phe atposition 71 with Tyr, and Tyr at position 87 with Phe are introduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, Gln at position 38 with Mg, Ala at position 43 with Thr, Pro atposition 44 with Val, and Tyr at position 87 with Phe are introduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, Gln at position 38 with Mg, Ala at position 43 with Thr, Pro atposition 44 with Val, and Phe at position 71 with Tyr are introduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, Val at position 15 with Leu, Pro at position 44 with Val, Phe atposition 71 with Tyr, and Tyr at position 87 with Phe are introduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, Val at position 15 with Leu, Ala at position 43 with Thr, Phe atposition 71 with Tyr, and Tyr at position 87 with Phe are introduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, Val at position 15 with Leu, Ala at position 43 with Thr, Pro atposition 44 with Val, and Tyr at position 87 with Phe are introduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, Val at position 15 with Leu, Ala at position 43 with Thr, Pro atposition 44 with Val, and Phe at position 71 with Tyr are introduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, Val at position 15 with Leu, Gln at position 38 with Arg, Phe atposition 71 with Tyr, and Tyr at position 87 with Phe are introduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, Val at position 15 with Leu, Gln at position 38 with Arg, Pro atposition 44 with Val, and Tyr at position 87 with Phe are introduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, Val at position 15 with Leu, Gln at position 38 with Arg, Pro atposition 44 with Val, and Phe at position 71 with Tyr are introduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, Val at position 15 with Leu, Gln at position 38 with Arg, Ala atposition 43 with Thr, and Tyr at position 87 with Phe are introduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, Val at position 15 with Leu, Gln at position 38 with Arg, Ala atposition 43 with Thr, and Phe at position 71 with Tyr are introduced,and

an amino acid sequence in which substitutions of Pro at position 8 withThr, Val at position 15 with Leu, Gln at position 38 with Arg, Ala atposition 43 with Thr, and Pro at position 44 with Val are introduced.

Specific examples of the amino acid sequence of VL in which fourmodifications are introduced in the amino acid sequence represented bySEQ ID NO:72 include the following amino acid sequences:

an amino acid sequence in which substitutions of Val at position 15 withLeu, Pro at position 44 with Val, Phe at position 71 with Tyr, and Tyrat position 87 with Phe are introduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, Ala at position 43 with Thr, Phe at position 71 with Tyr, and Tyrat position 87 with Phe are introduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, Gln at position 38 with Arg, Phe at position 71 with Tyr, and Tyrat position 87 with Phe are introduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, Val at position 15 with Leu, Phe at position 71 with Tyr, and Tyrat position 87 with Phe are introduced, and the like.

Specific examples of the amino acid sequence of VL in which threemodifications are introduced in the amino acid sequence represented bySEQ ID NO:72 may include the following amino acid sequences:

an amino acid sequence in which substitutions of Pro at position 8 withThr, Val at position 15 with Leu, and Phe at position 71 with Tyr areintroduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, Val at position 15 with Leu, and Tyr at position 87 with Phe areintroduced, an amino acid sequence in which substitutions of Pro atposition 8 with Thr, Phe at position 71 with Tyr, and Tyr at position 87with Phe are introduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, Gln at position 38 with Arg, and Phe at position 71 with Tyr areintroduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, Gln at position 38 with Arg, and Tyr at position 87 with Phe areintroduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, Ala at position 43 with Thr, and Phe at position 71 with Tyr areintroduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, Ala at position 43 with Thr, and Tyr at position 87 with Phe areintroduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, Pro at position 44 with Val, and Phe at position 71 with Tyr areintroduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, Pro at position 44 with Val, and Tyr at position 87 with Phe areintroduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, Phe at position 71 with Tyr, and Tyr at position 87 with Phe areintroduced,

an amino acid sequence in which substitutions of Gln at position 38 withArg, Phe at position 71 with Tyr, and Tyr at position 87 with Phe areintroduced,

an amino acid sequence in which substitutions of Ala at position 43 withThr, Phe at position 71 with Tyr, and Tyr at position 87 with Phe areintroduced,

an amino acid sequence in which substitutions of Pro at position 44 withVal, Phe at position 71 with Tyr, and Tyr at position 87 with Phe areintroduced, and the like.

Specific examples of the amino acid sequence of VL in which twomodifications are introduced in the amino acid sequence represented bySEQ ID NO:72 include the following amino acid sequences:

an amino acid sequence in which substitutions of Pro at position 8 withThr, and Val at position 15 with Leu are introduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, and Gln at position 38 with Arg are introduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, and Ala at position 43 with Thr are introduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, and Pro at position 44 with Val are introduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, and Phe at position 71 with Tyr are introduced,

an amino acid sequence in which substitutions of Pro at position 8 withThr, and Tyr at position 87 with Phe are introduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, and Gln at position 38 with Arg are introduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, and Ala at position 43 with Thr are introduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, and Pro at position 44 with Val are introduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, and Phe at position 71 with Tyr are introduced,

an amino acid sequence in which substitutions of Val at position 15 withLeu, and Tyr at position 87 with Phe are introduced,

an amino acid sequence in which substitutions of Gln at position 38 withArg, and Ala at position 43 with Thr are introduced,

an amino acid sequence in which substitutions of Gln at position 38 withArg, and Pro at position 44 with Val are introduced,

an amino acid sequence in which substitutions of Gln at position 38 withArg, and Phe at position 71 with Tyr are introduced,

an amino acid sequence in which substitutions of Gln at position 38 withArg, and Tyr at position 87 with Phe are introduced,

an amino acid sequence in which substitutions of Ala at position 43 withThr, and Pro at position 44 with Val are introduced,

an amino acid sequence in which substitutions of Ala at position 43 withThr, and Phe at position 71 with Tyr are introduced,

an amino acid sequence in which substitutions of Ala at position 43 withThr, and Tyr at position 87 with Phe are introduced,

an amino acid sequence in which substitutions of Pro at position 44 withVal, and Phe at position 71 with Tyr are introduced,

an amino acid sequence in which substitutions of Pro at position 44 withVal, and Tyr at position 87 with Phe are introduced, and

an amino acid sequence in which substitutions of Phe at position 71 withTyr, and Tyr at position 87 with Phe are introduced.

Specific examples of the amino acid sequence of VL in which onemodification is introduced in the amino acid sequence represented by SEQID NO:72 may include the following amino acid sequences:

an amino acid sequence in which a substitution of Pro at position 8 withThr is introduced,

an amino acid sequence in which a substitution of Val at position 15with Leu is introduced,

an amino acid sequence in which a substitution of Gln at position 38with Arg is introduced,

an amino acid sequence in which a substitution of Ala at position 43with Thr is introduced,

an amino acid sequence in which a substitution of Pro at position 44with Val is introduced,

an amino acid sequence in which a substitution of Phe at position 71with Tyr is introduced, and

an amino acid sequence in which a substitution of Tyr at position 87with Phe is introduced.

In addition, specific examples of the humanized antibody of the presentinvention include a humanized antibody wherein VII of the antibodycomprises the amino acid sequence represented by SEQ ID NO:71 and/or VLof the antibody comprises the amino acid sequence represented by SEQ IDNO:72, a humanized antibody wherein VH of the antibody comprises theamino acid sequence represented by SEQ ID NO:76 and/or VL of theantibody comprises the amino acid sequence represented by SEQ ID NO:72,a humanized antibody wherein VH of the antibody comprises the amino acidsequence represented by SEQ ID NO:78 and/or VL of the antibody comprisesthe amino acid sequence represented by SEQ ID NO:72, a humanizedantibody wherein VH of the antibody comprises the amino acid sequencerepresented by SEQ ID NO:80 and/or VL of the antibody comprises theamino acid sequence represented by SEQ ID NO:72, a humanized antibodywherein VH of the antibody comprises the amino acid sequence representedby SEQ ID NO:82 and/or VL of the antibody comprises the amino acidsequence represented by SEQ ID NO:72, a humanized antibody wherein VH ofthe antibody comprises the amino acid sequence represented by SEQ IDNO:71 and/or VL of the antibody comprises the amino acid sequencerepresented by SEQ ID NO:84, a humanized antibody wherein VH of theantibody comprises the amino acid sequence represented by SEQ ID NO:76and/or VL of the antibody comprises the amino acid sequence representedby SEQ ID NO:84, a humanized antibody wherein VH of the antibodycomprises the amino acid sequence represented by SEQ ID NO:78 and/or VLof the antibody comprises the amino acid sequence represented by SEQ IDNO:84, a humanized antibody wherein VH of the antibody comprises theamino acid sequence represented by SEQ ID NO:80 and/or VL of theantibody comprises the amino acid sequence represented by SEQ ID NO:84,a humanized antibody wherein VH of the antibody comprises the amino acidsequence represented by SEQ ID NO:82 and/or VL of the antibody comprisesthe amino acid sequence represented by SEQ ID NO:84 and the like.

A human antibody is originally an antibody naturally existing in thehuman body, and it also includes an antibody obtained from a humanantibody phage library or a human antibody-producing transgenic animal,which is prepared based on the recent advanced techniques in geneticengineering, cell engineering and developmental engineering.

The antibody existing in the human body can be prepared, for example byisolating a human peripheral blood lymphocyte, immortalizing it byinfecting with EB virus or the like, and then cloning it to therebyobtain lymphocytes capable of producing the antibody, culturing thelymphocytes thus obtained, and purifying the antibody from thesupernatant of the culture.

The human antibody phage library is a library in which antibodyfragments such as Fab and scFv are expressed on the phage surface byinserting a gene encoding an antibody prepared from a human B cell intoa phage gene. A phage expressing an antibody fragment having the desiredantigen binding activity can be recovered from the library, using itsactivity to bind to an antigen-immobilized substrate as the index. Theantibody fragment can be converted further into a human antibodymolecule comprising two full H chains and two full L chains by geneticengineering techniques.

A human antibody-producing transgenic animal is an animal in which ahuman antibody gene is integrated into cells. Specifically, a humanantibody-producing transgenic animal can be prepared by introducing agene encoding a human antibody into a mouse ES cell, grafting the EScell into an early stage embryo of other mouse and then developing it. Ahuman antibody is prepared from the human antibody-producing transgenicnon-human animal by obtaining a human antibody-producing hybridoma by ahybridoma preparation method usually carried out in non-human mammals,culturing the obtained hybridoma and forming and accumulating the humanantibody in the supernatant of the culture.

In the amino acid sequence constituting the above antibody or antibodyfragment, an antibody or antibody fragment thereof in which one or moreamino acids are deleted, substituted, inserted or added, having activitysimilar to the above antibody or antibody fragment is also included inthe antibody or antibody fragment of the present invention.

The number of amino acids which are deleted, substituted, insertedand/or added is one or more, and is not specifically limited, but it iswithin the range where deletion, substitution or addition is possible byknown methods such as the site-directed mutagenesis described inMolecular Cloning, Second Edition, Cold Spring Harbor Laboratory Press(1989); Current Protocols in Molecular Biology, John Wiley & Sons(1987-1997); Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad.Sci. USA, 79, 6409 (1982); Gene, 34, 315 (1985), Nucleic Acids Research,13, 4431 (1985); Proc. Natl. Acad Sci. USA, 82, 488 (1985) or the like.For example, the number is 1 to dozens, preferably 1 to 20, morepreferably 1 to 10, and most preferably 1 to 5.

The expression “one or more amino acid residue(s) is/are deleted,substituted, inserted and/or added” in the amino acid sequence of theabove antibody means the followings. That is, it means there isdeletion, substitution, insertion or addition of one or plural aminoacids at optional positions in the same sequence and in one or pluralamino acid sequences. Also, the deletion, substitution, insertion oraddition may occur at the same time and the amino acid which issubstituted, inserted or added may be either a natural type or anon-natural type. The natural type amino acid includes L-alanine,L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine,L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan,L-tyrosine, L-valine, L-cysteine and the like.

Preferable examples of mutually substitutable amino acids are shownbelow. The amino acids in the same group are mutually substitutable.

Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine,2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine,t-butylalanine, cyclohexylalanineGroup B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamicacid, 2-aminoadipic acid, 2-aminosuberic acidGroup C: asparagine, glutamineGroup D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid,2,3-diaminopropionic acidGroup E: proline, 3-hydroxyproline, 4-hydroxyprolineGroup F: serine, threonine, homoserineGroup G: phenylalanine, tyrosine

The antibody fragment of the present invention includes Fab, F(ab′)₂,Fab′, scFv, diabody, dsFv, a peptide comprising CDR and the like.

An Fab is an antibody fragment having a molecular weight of about 50,000and having antigen binding activity, in which about a half of theN-terminal side of H chain and the entire L chain, among fragmentsobtained by treating an IgG antibody molecule with a protease, papain(cleaved at an amino acid residue at position 224 of the H chain), arebound together through a disulfide bond. The Fab of the presentinvention can be produced by treating a monoclonal antibody whichspecifically recognizes three-dimensional structure of an extracellularregion of ASCT2 and binds to the extracellular region with papain. Also,the Fab can be produced by inserting DNA encoding Fab of the antibodyinto an expression vector for prokaryote or an expression vector foreukaryote, and introducing the vector into a prokaryote or eukaryote toexpress the Fab.

An F(ab′)₂ is an antibody fragment having a molecular weight of about100,000 and having antigen binding activity and comprising two Fabregions which are bound in the hinge position obtained by digesting thelower part of two disulfide bonds in the hinge region of IgG with anenzyme, pepsin. The F(ab′)₂ of the present invention can be produced bytreating a monoclonal antibody which specifically recognizesthree-dimensional structure of an extracellular region of ASCT2 andbinds to the extracellular region with pepsin. Also, the F(ab′)₂ can beproduced by binding Fab′ described below via a thioether bond or adisulfide bond.

An Fab′ is an antibody fragment having a molecular weight of about50,000 and antigen binding activity, which is obtained by cleaving adisulfide bond at the hinge region of the above F(ab′)₂. The Fab′ of thepresent invention can be produced by F(ab′)₂ which specificallyrecognizes three-dimensional structure of an extracellular region ofASCT2 and binds to the extracellular region with a reducing agent, suchas dithiothreitol. Also, the Fab′ can be produced by inserting DNAencoding Fab′ fragment of the antibody into an expression vector forprokaryote or an expression vector for eukaryote, and introducing thevector into a prokaryote or eukaryote to express the Fab′.

An scFv is a VH-P-VL or VL-P-VH polypeptide in which one chain VH andone chain VL are linked using an appropriate peptide linker (hereinafterreferred to as “P”) and is an antibody fragment having antigen bindingactivity. The scFv of the present invention can be produced by obtainingcDNAs encoding VH and VL of a monoclonal antibody which specificallyrecognizes three-dimensional structure of an extracellular region ofASCT2 and binds to the extracellular region, constructing DNA encodingscFv, inserting the DNA into an expression vector for prokaryote or anexpression vector for eukaryote, and then introducing the expressionvector into a prokaryote or eukaryote to express the scFv.

A diabody is an antibody fragment wherein scFv is dimerized, is anantibodt fragment having divalent antigen binding activity. In thedivalent antigen binding activity, two antigens may be the same ordifferent. The diabody of the present invention can be produced byobtaining cDNAs encoding VH and VL of a monoclonal antibody whichspecifically recognizes three-dimensional structure of an extracellularregion of ASCT2 and binds to the extracellular region, constructing DNAencoding scFv so that the length of the amino acid sequence of thepeptide linker is 8 or less residues, inserting the DNA into anexpression vector for prokaryote or an expression vector for eukaryote,and then introducing the expression vector into a prokaryote oreukaryote to express the diabody.

A dsFv is obtained by binding polypeptides in which one amino acidresidue of each of VH and VL is substituted with a cysteine residue viaa disulfide bond between the cysteine residues. The amino acid residueto be substituted with a cysteine residue can be selected based on athree-dimensional structure estimation of the antibody in accordancewith a known methods [Protein Engineering, 7, 697 (1994)]. The dsFv ofthe present invention can be produced by obtaining cDNAs encoding VH andVL of a monoclonal antibody which specifically recognizesthree-dimensional structure of an extracellular region of ASCT2 andbinds to the extracellular region, constructing DNA encoding dsFv,inserting the DNA into an expression vector for prokaryote or anexpression vector for eukaryote, and then introducing the expressionvector into a prokaryote or eukaryote to express the dsFv.

A peptide comprising CDR is constituted by including one or more regionsof CDRs of VH or VL. Peptide comprising plural CDRs can be bounddirectly or via an appropriate peptide linker. The peptide comprisingCDR of the present invention can be produced by constructing DNAencoding CDRs of VH and VL of a monoclonal antibody which specificallyrecognizes three-dimensional structure of an extracellular region ofASCT2 and binds to the extracellular region, inserting the DNA into anexpression vector for prokaryote or an expression vector for eukaryote,and then introducing the expression vector into a prokaryote oreukaryote to express the peptide. The peptide comprising CDR can also beproduced by a chemical synthesis method such as Fmoc method or tBocmethod.

The monoclonal antibody of the present invention includes an antibodyconjugate in which a monoclonal antibody or an antibody fragment thereofwhich specifically recognizes a three-dimensional structure of anextracellular region of ASCT2 and binds to the extracellular region ischemically or genetically bound to a radioisotope, an agent having a lowmolecular weight, an agent having a high molecular weight, a protein, atherapeutic antibody or the like.

The antibody conjugate of the present invention can be produced bychemically conjugating a radioisotope, an agent having a low molecularweight, an agent having a high molecular weight, a protein, atherapeutic antibody or the like to the N-terminal side or C-terminalside of an H chain or an L chain of the monoclonal antibody or theantibody fragment thereof, an appropriate substituent or side chain ofthe antibody or the antibody fragment, a sugar chain in the antibody orthe antibody fragment or the like, which specifically recognizes athree-dimensional structure of an extracellular region of ASCT2 andbinds to the extracellular region in the present invention [AntibodyEngineering Handbook, published by Chijin Shokan (1994)].

Also, the antibody conjugate can be genetically produced by linking aDNA encoding the monoclonal antibody or the antibody fragment thereofwhich specifically recognizes three-dimensional structure of anextracellular region of ASCT2 and binds to the extracellular region inthe present invention to other DNA encoding a protein or a therapeuticantibody to be conjugated, inserting the DNA into a vector forexpression, and introducing the expression vector into an appropriatehost cell.

The radioisotope includes ¹³¹I, ¹²⁵I, ⁹⁰Y, ⁶⁴Cu, ¹⁹⁹Tc, ⁷⁷Lu, ²¹¹At andthe like. The radioisotope can directly be conjugated with the antibodyby Chloramine-T method or the like. Also, a substance chelating theradioisotope can be conjugated with the antibody. The chelating agentincludes 1-isothiocyanatobenzyl-3-methyldiethylene-triaminepentaaceticacid (MX-DTPA) and the like.

The agent having a low molecular weight includes an anti-tumor agentsuch as an alkylating agent, a nitrosourea agent, a metabolismantagonist, an antibiotic substance, an alkaloid derived from a plant, atopoisomerase inhibitor, an agent for hormonotherapy, a hormoneantagonist, an aromatase inhibitor, a P glycoprotein inhibitor, aplatinum complex derivative, an M-phase inhibitor and a kinase inhibitor[Rinsho Syuyo-gaku (Clinical Oncology), Gan to Kagaguryoho-Sha (1996)],a steroid agent such as hydrocortisone and prednisone, a nonsteroidalagent such as aspirin and indomethacin, immune-regulating agent such asaurothiomalate, penicillamine, immuno-suppressing agent such ascyclophosphamide and azathioprine, anti-inflammatory agent such asanti-histamine agent, for example, chlorpheniramine maleate andclemastine [Ensho to Kouensho-Ryoho (Inflammation and Anti-inflammationTherapy), Ishiyaku Shuppann (1982)] and the like. Examples of theantitumor agent include amifostine (Ethyol), cisplatin, dacarbazine(DTIC), dactinomycin, mecloretamin (nitrogen mustard), streptozocin,cyclophosphamide, iphosphamide, carmustine (BCNU), lomustine (CCNU),doxorubicin (adriamycin), epirubicin, gemcitabine (Gemsal),daunorubicin, procarbazine, mitomycin, cytarabine, etoposide,methotrexate, 5-fluorouracil, fluorouracil, vinblastine, vincristine,bleomycin, daunomycin, peplomycin, estramustine, paclitaxel (Taxol),docetaxel (Taxotea), aldesleukin, asparaginase, busulfan, carboplatin,oxaliplatin, nedaplatin, cladribine, camptothecin,10-hydroxy-7-ethylcamptothecin (SN38), floxuridine, fludarabine,hydroxyurea, iphosphamide, idarubicin, mesna, irinotecan (CPT-11),nogitecan, mitoxantrone, topotecan, leuprolide, megestrol, melfalan,mercaptopurine, hydroxycarbamide, plicamycin, mitotane, pegasparagase,pentostatin, pipobroman, streptozocin, tamoxifen, goserelin,leuprorelin, flutamide, teniposide, testolactone, thioguanine, thiotepa,uracil mustard, vinorelbine, chlorambucil, hydrocortisone, prednisolone,methylprednisolone, vindesine, nimustine, semustine, capecitabine,Tomudex, azacytidine, UFT, oxaliplatin, gefitinib (Iressa), imatinib(STI 571), elrotinib, FMS-like tyrosine kinase 3 (Flt3) inhibitor,vascular endothelial growth facotr receptor (VEGFR) inhibitor,fibroblast growth factor receptor (FGFR) inhibitor, epidermal growthfactor receptor (EGFR) inhibitor such as Iressa and Tarceva, radicicol,17-allylamino-17-demethoxygeldanamycin, rapamycin, amsacrine,all-trans-retinoic acid, thalidomide, lenalidomide, anastrozole,fadrozole, letrozole, exemestane, gold thiomalate, D-penicillamine,bucillamine, azathioprine, mizoribine, cyclosporine, rapamycin,hydrocortisone, bexarotene (Targretin), tamoxifen, dexamethasone,progestin substances, estrogen substances, anastrozole (Arimidex),Leuplin, aspirin, indomethacin, celecoxib, azathioprine, penicillamine,gold thiomalate, chlorpheniramine maleate, chlorpheniramine, clemastine,tretinoin, bexarotene, arsenic, voltezomib, allopurinol, calicheamicin,ibritumomab tiuxetan, Targretin, ozogamine, clarithromycin, leucovorin,ifosfamide, ketoconazole, aminoglutethimide, suramin, methotrexate,maytansinoid and derivatives thereof.

The method for conjugating the agent having low molecular weight withthe antibody includes a method in which the agent and an amino group ofthe antibody are conjugated through glutaraldehyde, a method in which anamino group of the agent and a carboxyl group of the antibody areconjugated through water-soluble carbodiimide, and the like.

The agent having a high molecular weight includes polyethylene glycol(hereinafter referred to as “PEG”), albumin, dextran, polyoxyethylene,styrene-maleic acid copolymer, polyvinylpyrrolidone, pyran copolymer,hydroxypropylmethacrylamide, and the like. By binding these compoundshaving a high molecular weight to an antibody or antibody fragment, thefollowing effects are expected: (1) improvement of stability againstvarious chemical, physical or biological factors, (2) remarkableprolongation of half life in blood, (3) disappearance of immunogenicity,suppression of antibody production, and the like [Bioconjugate Drug,Hirokawa Shoten (1993)]. For example, the method for binding. PEG to anantibody includes a method in which an antibody is allowed to react witha PEG-modifying reagent [Bioconjugate Drug, Hirokawa Shoten (1993)]. ThePEG-modifying reagent includes a modifying agent of ε-amino group oflysine (Japanese Published Unexamined Patent Application No. 178926/86),a modifying agent of a carboxyl group of aspartic acid and glutamic acid(Japanese Published Unexamined Patent Application No. 23587/81), amodifying agent of a guanidino group of arginine (Japanese PublishedUnexamined Patent Application No. 117920/90) and the like.

The immuno stimulator may be any natural products known asimmunoadjuvants. Examples of an agent enhancing immunogen includeβ(1→3)glucan (lentinan, schizophyllan), α-galactosylceramide (KRN7000)and the like.

The protein includes a cytokine or a growth factor which activates aimmunocompetent cell, such as NK cell, macrophage or neutrophil, a toxicprotein, and the like.

Examples of the cytokine or the growth factor include interferon(hereinafter referred to as “INF”)-α, INF-β, INF-γ, interleukin(hereinafter referred to as “IL”)-2, IL-12, IL-15, IL-18, IL-21, IL-23,granulocyte-colony stimulating factor (G-CSF), granulocytemacrophage-colony stimulating factor (GM-CSF), macrophage-colonystimulating factor (M-CSF) and the like. The toxic protein includesricin, diphtheria toxin, ONTAK and the like, and also includes a toxicprotein wherein mutation is introduced into a protein in order tocontrol the toxicity.

The therapeutic antibody includes an antibody against an antigen inwhich apoptosis is induced by binding of the antibody, an antibodyagainst an antigen participating in formation of pathologic state oftumor, an antibody which regulates immunological function and anantibody relating to angiogenesis in the pathologic part.

The antigen in which apoptosis is induced by binding of the antibodyincludes cluster of differentiation (hereinafter “CD”) 19, CD20, CD21,CD22, CD23, CD24, CD37, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77,CDw78, CD79a, CD79b, CD80 (B7.1), CD81, CD82, CD83, CDw84, CD85, CD86(B7.2), human leukocyte antigen (HLA)-Class II, epidermal growth factorreceptor (EGFR) and the like.

The antigen participating in formation of pathologic state of tumor orthe antigen for the antibody which regulates immunological functionincludes CD4, CD40, CD40 ligand, B7 family molecule (CD80, CD86, CD274,B7-DC, B7-H2, B7-H3, B7-H4), ligand of B7 family molecule (CD28, CTLA-4,ICOS, PD-1, BTLA), OX-40, OX-40 ligand, CD137, tumor necrosis factor(TNF) receptor family molecule (DR4, DRS, TNFR1, TNFR2), TNF-relatedapoptosis-inducing ligand receptor (TRAIL) family molecule, receptorfamily of TRAIL family molecule (TRAIL-R1, TRAIL-R2, TRAIL-R3,TRAIL-R4), receptor activator of nuclear factor kappa B ligand (RANK),RANK ligand, CD25, folic acid receptor 4, cytokine [IL-10, IL-4, IL-5,IL-6, IL-10, IL-13, transforming growth factor (TGF) β, TNFα, etc.],receptors of these cytokines, chemokine (SLC, ELC, 1-309, TARC, MDC,CTACK, etc.) and receptors of these chemokines.

The antigen for the antibody which inhibits angiogenesis in thepathologic part includes vascular endothelial growth factor (VEGF),angiopoietin, fibroblast growth factor (FGF), EGF, platelet-derivedgrowth factor (PDGF), insulin-like growth factor (IGF), erythropoietin(EPO), TGFβ, IL-8, Ephilin, SDF-1, receptors thereof and the like.

A fusion antibody with a protein or therapeutic antibody can be producedby linking a cDNA encoding a monoclonal antibody or antibody fragment toa cDNA encoding the protein, constructing a DNA encoding the fusionantibody, inserting the DNA into an expression vector for prokaryote oreukaryote, and then introducing the expression vector into a prokaryoteor eukaryote to express the fusion antibody.

In the case where the above antibody conjugate is used for the detectionmethod, method for quantitative determination, detection reagent,reagent for quantitative determination or diagnostic agent in thepresent invention, examples of the agent to which a monoclonal antibodyor an antibody fragment thereof which specifically recognizes athree-dimensional structure of an extracellular region of ASCT2 andbinds to the extracellular region is bound includes a label used inroutine immunological detecting or measuring method. The label includesenzymes such as alkaline phosphatase, peroxidase and luciferase,luminescent materials such as acridinium ester and lophine, fluorescentmaterials such as fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (RITC), and the like.

Furthermore, the present invention includes a monoclonal antibody whichinhibits intracellular uptake of amino acids by ASCT2, and an antibodyfragment thereof.

Examples of the method of evaluating an inhibitory activity of theantibody or antibody fragment thereof of the present invention onintracellular uptake of amino acids by ASCT2, include a method in whichan antibody or an antibody fragment thereof is allowed to react with anormal or cancer cell expressing ASCT2, and then is evaluated theinhibition of glutamine-dependent proliferation using a viable cellcounting reagent, or the like [J. Surgical Research, 90, 149 (2000)], amethod in which an antibody or an antibody fragment thereof is allowedto react with a normal or cancer cell expressing ASCT2, and then isevaluated the inhibition of uptake of amino acids such as radioactivematerial-labeled alanine using appropriate equipment such asscintillation counter [J. Biol. Chem., 271, 14883 (1996)], or the like.

In addition, the present invention includes a monoclonal antibody and anantibody fragment thereof having a cellular cytotoxicity such as acomplement-dependent cytotoxicity (CDC) activity, or anantibody-dependent cellular cytotoxicity (ADCC) activity.

The CDC activity or ADCC activity of the antibody or the antibodyfragment thereof of the present invention against the antigen-positivecell line can be evaluated by known assay methods [Cancer Immunol.Immunother, 36, 373 (1993)].

Furthermore, the present invention includes a monoclonal antibody havingan apoptosis-inducing activity, and an antibody fragment thereof.

Moreover, the present invention includes a monoclonal antibody whichdoes not bind to mouse ASCT2, but also binds to human ASCT2, and anantibody fragment thereof.

Further, the present invention includes a monoclonal antibody whichbinds to at least the EL2 region of human ASCT2, and an antibodyfragment thereof, for example, a monoclonal antibody which binds to atleast any one of amino acids at positions 154, 159 to 160, 163 to 171,173 to 174, 177, 188, 204 to 205, 207, 210 to 212, and 214 to 223, inthe amino acid sequence represented by SEQ ID NO:2, and an antibodyfragment thereof.

The binding specificity of the antibody or antibody fragment thereof ofthe present invention can be evaluated by a known epitope analysismethod, for example by measuring the binding activity to human/mousechimeric ASCT2 in which an appropriate site is replaced with a sequenceof mouse ASCT2, based on the amino acid sequence information.

Also, the present invention relates to a therapeutic agent for a diseaserelating to ASCT2 comprising the monoclonal antibody or antibodyfragment which specifically recognizes three-dimensional structure of anextracellular region of ASCT2 and binds to the extracellular region asan active ingredient.

The disease relating to ASCT2 is not limited, so long as it is a diseaserelating to a cell expressing ASCT2, such as cancer.

The cancer includes blood cancer, breast cancer, uterine cancer,colorectal cancer, esophageal cancer, stomach cancer, ovarian cancer,lung cancer, renal cancer, rectal cancer, thyroid cancer, uterine cervixcancer, small intestinal cancer, prostate cancer and pancreatic cancer.Preferable examples of the cancer include blood cancer, esophagealcancer, stomach cancer, colorectal cancer, liver cancer and prostatecancer. Examples of blood cancer include myeloid leukemia, lymphoidleukemia, multiple myeloma, Hodgkin's lymphoma and non-Hodgkin'slymphoma.

The therapeutic agent in the present invention includes a therapeuticagent comprising the above monoclonal antibody or antibody fragment ofthe present invention as an active ingredient.

The therapeutic agent comprising the antibody or antibody fragmentthereof, or conjugate thereof of the present invention may comprise onlythe antibody or antibody fragment thereof, or conjugate thereof as anactive ingredient. It is generally preferred that the therapeutic agentis prepared as a pharmaceutical preparation produced by an appropriatemethod well known in the technical field of pharmaceutics, and by mixingit with one or more pharmaceutically acceptable carriers.

It is preferred to administer the therapeutic agent by the route that ismost effective for the treatment. Examples include oral administrationand parenteral administration, such as buccal, tracheal, rectal,subcutaneous, intramuscular or intravenous administration andintravenous administration is preferred.

The therapeutic agent may be in the form of spray, capsules, tablets,powder, granules, syrup, emulsion, suppository, injection, ointment,tape, and the like.

Although the dose or the frequency of administration varies depending onthe objective therapeutic effect, administration method, treatingperiod, age, body weight and the like, it is usually 10 μg/kg to 8 mg/kgper day and per adult.

Further, the present invention relates to a method for immunologicallydetecting or measuring ASCT2, a reagent for immunologically detecting ormeasuring ASCT2, a method for immunologically detecting or measuring acell expressing ASCT2, and a diagnostic agent for diagnosing a diseaserelating to ASCT2, comprising a monoclonal antibody which specificallyrecognizes a native three-dimensional structure of an extracellularregion of ASCT2 and binds to the extracellular region, or an antibodyfragment thereof as an active ingredient.

In the present invention, the method for detecting or measuring theamount of ASCT2 may be any known method. For example, it includes animmunological detecting or measuring method.

The immunological detecting or measuring method is a method in which anantibody amount or an antigen amount is detected or determined using alabeled antigen or antibody. Examples of the immunological detecting ormeasuring method include radioactive substance-labeled immunoantibodymethod (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay(FIA), luminescent immunoassay, Western blotting method,physico-chemical method and the like.

The above disease relating to ASCT2 can be diagnosed by detecting ormeasuring a cell expressing ASCT2 by using the monoclinal antibody orantibody fragment of the present invention.

For the detection of the cell expressing ASCT2, known immunologicaldetection methods can be used, and an immunoprecipitation method, afluorescent cell staining method, an immune tissue staining method andthe like are preferably used. Also, a fluorescent antibody stainingmethod using FMAT 8100 HTS system (Applied Biosystem) and the like canbe used.

In the present invention, the living body sample to be used fordetecting or measuring ASCT2 is not particularly limited, so long as ithas a possibility of containing a cell expressing ASCT2, such as tissuecells, blood, blood plasma, serum, pancreatic fluid, urine, fecalmatter, tissue fluid or culture fluid.

The diagnostic agent containing the monoclonal antibody or antibodyfragment thereof, or conjugate thereof may further contain a reagent forcarrying out an antigen-antibody reaction or a reagent for detection ofthe reaction depending on the desired diagnostic method. The reagent forcarrying out the antigen-antibody reaction includes a buffer, a salt,and the like. The reagent for detection includes a reagent generallyused for the immunological detecting or measuring method, such aslabeled secondary antibody which recognizes the monoclonal antibody,antibody fragment thereof or conjugates thereof and substratecorresponding to the labeling.

The present invention can provide a monoclonal antibody whichspecifically recognizes a native three-dimensional structure of anextracellular region of system ASC amino acid trasporter 2 (hereinafterreferred to as ASCT2) and binds to the extracellular region, or anantibody fragment thereof; a hybridoma which produces the antibody; aDNA which encodes the antibody; a vector which contains the DNA; atransformant obtainable by introducing the vector; a process forproducing an antibody or an antibody fragment thereof using thehybridoma or the transformant; and a therapeutic agent using theantibody or the antibody fragment thereof, and a diagnostic agent usingthe antibody or the antibody fragment thereof.

A process for producing the antibody of the present invention, a methodfor treating the disease and a method for diagnosing the disease arespecifically described below.

1. Preparation Method of Monoclonal Antibody (1) Preparation of Antigen

ASCT2 or a cell expressing ASCT2 as an antigen can be obtained byintroducing an expression vector comprising cDNA encoding a full lengthof ASCT2 or a partial length thereof is introduced into Escherichiacoli, yeast, an insect cell, an animal cell or the like. In addition,ASCT2 can be purified from various human tumor cell lines, human tissueand the like which express a large amount of ASCT2. The tumor cell lineand the tissue can be allowed to use as antigens. Furthermore, asynthetic peptide having a partial sequence of the ASCT2 can be preparedby a chemical synthesis method such as Fmoc method or tBoc method andused as an antigen.

ASCT2 used in the present invention can be produced, for example, byexpressing a DNA encoding ASCT2 in a host cell using a method describedin Molecular Cloning, A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press (1989), Current Protocols in Molecular Biology,John Wiley & Sons (1987-1997) or the like according to the followingmethod.

Firstly, a recombinant vector is prepared by inserting a full lengthcDNA comprising the region encoding ASCT2 into downstream of a promoterof an appropriate expression vector. At this time, if necessary, a DNAfragment having an appropriate length containing a region encoding thepolypeptide based on the full length cDNA, and the DNA fragment may beused instead of the above full length cDNA. Next, a transformantproducing ASCT2 can be obtained by introducing the recombinant vectorinto a host cell suitable for the expression vector.

The expression vector may be any one, so long as it can replicateautonomously in the host cell to be used or it can be integrated into achromosome comprising an appropriate promoter at such a position thatthe DNA encoding the polypeptide can be transcribed.

The host cell may be any one, so long as it can express the objectivegene. Examples include a microorganism which belongs to the generaEscherichia, such as Escherichia coli, yeast, an insect cell, an animalcell and the like.

When a prokaryote such as Escherichia coli is used as the host cell, itis preferred that the recombinant vector used in the present inventionis autonomously replicable in the prokaryote and comprising a promoter,a ribosome binding sequence, the DNA encoding ASCT2 and a transcriptiontermination sequence. The recombinant vector is not necessary to have atranscription termination sequence, but a transcription terminationsequence is preferably set just below the structural gene. Therecombinant vector may further comprise a gene regulating the promoter.

Also, the above recombinant vector is preferably a plasmid in which thespace between Shine-Dalgarno sequence, which is the ribosome bindingsequence, and the initiation codon is adjusted to an appropriatedistance (for example, 6 to 18 nucleotides).

Furthermore, the nucleotide sequence of the DNA encoding ASCT2 can besubstituted with another base so as to be a suitable codon forexpressing in a host cell, thereby improve the productivity of theobjective ASCT2.

Any expression vector can be used, so long as it can perform in the hostcell to be used. Examples of the expression vector includes pBTrp2,pBTac1, pBTac2 (all manufactured by Roche Diagnostics), pKK233-2(manufactured by Pharmacia), pSE280 (manufactured by Invitrogen),pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured by QIAGEN),pKYP10 (Japanese Published Unexamined Patent Application No. 110600/83),pKYP200 [Agricultural Biological Chemistry, 48, 669 (1984)], pLSA1[Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA,82, 4306 (1985)], pBluescript II SK(−) (manufactured by Stratagene),pTrs30 [prepared from Escherichia coli JM109/pTrS30 (FERM BP-5407)],pTrs32 [prepared from Escherichia coli JM109/pTrS32 (FERM BP-5408)],pGHA2 [prepared from Escherichia coli IGHA2 (FERM BP-400), JapanesePublished Unexamined Patent Application No. 221091/85], pGKA2 [preparedfrom Escherichia coli IGKA2 (FERM BP-6798), Japanese PublishedUnexamined Patent Application No. 221091/85], pTerm2 (U.S. Pat. No.4,686,191, U.S. Pat. No. 4,939,094, U.S. Pat. No. 5,160,735), pSupex,pUB110, pTP5, pC194, pEG400 [J. Bacteriol., 172, 2392 (1990)], pGEX(manufactured by Pharmacia), pET system (manufactured by Novagen),pME18SFL3 and the like.

Any promoter can be used, so long as it can function in the host cell tobe used. Examples include promoters derived from Escherichia coli, phageand the like, such as trp promoter (Ptrp), lac promoter, PL promoter, PRpromoter and T7 promoter. Also, artificially designed and modifiedpromoters, such as a promoter in which two Ptrp are linked in tandem,tac promoter, lacT7 promoter and letl promoter, can be used.

Examples of host cell include Escherichia coli XL1-Blue, Escherichiacoli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000,Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109,Escherichia coli BB 101, Escherichia coli No. 49, Escherichia coliW3110, Escherichia coli NY49, Escherichia coli DH5α and the like.

Any introduction method of the recombinant vector can be used, so longas it is a method for introducing DNA into the host cell, and examplesinclude a method using a calcium ion described in Proc. Natl. Acad. Sci.USA, 69, 2110 (1972), Gene, 17, 107 (1982) and Molecular & GeneralGenetics, 168, 111 (1979) and the like.

When an animal cell is used as the host cell, any expression vector canbe used, so long as it can function in the animal cell. Examples includepcDNAL pcDM8 (manufactured by Funakoshi), pAGE107 [Japanese PublishedUnexamined Patent Application No. 22979/91; Cytotechnology, 3, 133(1990)], pAS3-3 (Japanese Published Unexamined Patent Application No.227075/90), pCDM8 [Nature, 329, 840,(1987)], pcDNAI/Amp (manufactured byInvitrogen), pcDNA3.1 (manufactured by Invitrogen), pREP4 (manufacturedby Invitrogen), pAGE103 Biochemistry, 101, 1307 (1987)1 pAGE210,pME18SFL3, pKANTEX93 (WO 97/10354) and the like.

Any promoter can be used, so long as it can function in an animal cell.Examples include a promoter of immediate early (IE) gene ofcytomegalovirus (CMV), SV40 early promoter, a promoter of retrovirus, ametallothionein promoter, a heat shock promoter, SRα promoter, Molonymurine leukemia virus promoter or enhancer, and the like. Also, theenhancer of the IE gene of human CMV can be used together with thepromoter.

The host cell includes human Namalwa cell, monkey COS cell, Chinesehamster ovary (CHO) cell, HST5637 (Japanese Published Unexamined PatentApplication No. 299/88) and the like.

Any introduction method of the recombinant vector can be used, so longas it is a method for introducing DNA into an animal cell, and examplesinclude electroporation [Cytotechnology, 3, 133 (1990)], the calciumphosphate method (Japanese Published Unexamined Patent Application No.227075/90), the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413(1987)], and the like.

ASCT2 can be produced by culturing the transformant derived from amicroorganism, an animal cell or the like having a recombinant vectorcomprising the DNA encoding ASCT2 in a medium to form and accumulateASCT2 in the culture, and recovering it from the culture. The method forculturing the transformant in the medium is carried out according to theusual method used in culturing of hosts.

When ASCT2 is expressed in a cell derived from eukaryote, ASCT2 to whichsugars or sugar chains bind can be obtained.

When a microorganism transformed with a recombinant vector containing aninducible promoter is cultured, an inducer can be added to the medium,if necessary. For example, isopropyl-β-D-thiogalactopyranoside or thelike can be added to the medium when a microorganism transformed with arecombinant vector using lac promoter is cultured; or indoleacrylic acidor the like can be added thereto when a microorganism transformed with arecombinant vector using trp promoter is cultured.

When a transformant obtained using an animal cell as the host cell iscultured, the medium includes generally used RPMI 1640 medium [TheJournal of the American Medical Association, 199, 519 (1967)], Eagle'sMEM medium [Science, 122, 501 (1952)], Dulbecco's modified MEM medium[Virology, 8, 396 (1959)] and 199 medium [Proceeding of the Society forthe Biological Medicine, 73, 1 (1950)], Iscoove's modified Dulbecco'smedium (IMDM), the media to which fetal calf serum, etc. is added, andthe like. The culturing is carried out generally at a pH of 6 to 8 and30 to 40° C. for 1 to 7 days in the presence of 5% CO₂. If necessary, anantibiotic such as kanamycin or penicillin can be added to the mediumduring the culturing.

Regarding the expression method of the gene encoding ASCT2, in additionto direct expression, secretory production, fusion protein expressionand the like can be carried out according to the method described inMolecular Cloning, A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press (1989).

The process for producing ASCT2 includes a method of intracellularexpression in a host cell, a method of extracellular secretion from ahost cell, a method of producing on a host cell membrane outer envelope,and the like. The appropriate method can be selected by changing thehost cell used and the structure of the ASCT2 produced.

When the ASCT2 is produced in a host cell or on a host cell membraneouter envelope, ASCT2 can be positively secreted extracellularly inaccordance with the method of Paulson et al. [J. Biol. Chem., 264, 17619(1989)], the method of Lowe et al. [Proc. Natl. Acad. Sci. USA, 86, 8227(1989), Genes Develop., 4, 1288 (1990)], the methods described inJapanese Published Unexamined Patent Application No. 336963/93 and WO94/23021, and the like.

Also, the production amount of ASCT2 can be increased in accordance withthe method described in Japanese Published Unexamined Patent ApplicationNo. 227075/90 utilizing a gene amplification system using adihydrofolate reductase gene.

The resulting ASCT2 can be isolated and purified, for example, asfollows.

When ASCT2 is intracellularly expressed in a dissolved state, the cellsafter culturing are recovered by centrifugation, suspended in an aqueousbuffer and then disrupted using ultrasonicator, French press, MantonGaulin homogenizer, dynomill or the like to obtain a cell-free extract.The cell-free extract is centrifuged to obtain a supernatant, and apurified preparation can be obtained by subjecting the supernatant to ageneral protein isolation and purification techniques such as solventextraction; salting out with ammonium sulfate etc.; desalting;precipitation with an organic solvent; anion exchange chromatographyusing a resin such as diethylaminoethyl (DEAE)-sepharose, DIAION HPA-75(manufactured by Mitsubishi Chemical); cation exchange chromatographyusing a resin such as S-Sepharose FF (manufactured by Pharmacia);hydrophobic chromatography using a resin such as butyl-Sepharose orphenyl-Sepharose; gel filtration using a molecular sieve; affinitychromatography; chromatofocusing; electrophoresis such as isoelectricfocusing; and the like which may be used alone or in combination.

When ASCT2 is expressed intracellularly by forming an inclusion body,the cells are recovered, disrupted and centrifuged in the same manner,and the inclusion body of ASCT2 are recovered as a precipitationfraction. The recovered inclusion body of the protein is solubilizedwith a protein denaturing agent. The protein is made into a normalthree-dimensional structure by diluting or dialyzing the solubilizedsolution, and then a purified preparation of ASCT2 is obtained by thesame isolation purification method as above.

When ASCT2 or the derivative such as a glycosylated product is secretedextracellularly, ASCT2 or the derivative such as a glycosylated productcan be recovered from the culture supernatant. That is, the culture istreated by a method such as centrifugation in the same manner as aboveto obtain a culture supernatant, a purified preparation of ASCT2 can beobtained from the culture supernatant by the same isolation purificationmethod as above.

Also, ASCT2 used in the present invention can be produced by a chemicalsynthesis method, such as Fmoc method or tBoc method. Also, it can bechemically synthesized using a peptide synthesizer manufactured byAdvanced ChemTech, Perkin-Elmer, Pharmacia, Protein TechnologyInstrument, Synthecell-Vega, PerSeptive, Shimadzu Corporation, or thelike.

(2) Immunization of Animal and Preparation of Antibody-Producing Cellfor Fusion

A mouse, rat or hamster 3 to 20 weeks old is immunized with the antigenprepared in the above (1), and antibody-producing cells are collectedfrom the spleen, lymph node or peripheral blood of the animal. Also,when the increase of a sufficient titer in the above animal isrecognized due to low immunogenecity, a ASCT2 knockout mouse may by usedas an animal to be immunized.

The immunization is carried out by administering the antigen to theanimal through subcutaneous, intravenous or intraperitoneal injectiontogether with an appropriate adjuvant (for example, complete Freund'sadjuvant, combination of aluminum hydroxide gel with pertussis vaccine,or the like). When the antigen is a partial peptide, a conjugate isproduced with a carrier protein such as BSA (bovine serum albumin), KLH(keyhole limpet hemocyanin) or the like, which is used as the antigen.

The administration of the antigen is carried out 5 to 10 times every oneweek or every two weeks after the first administration. On the 3rd to7th day after each administration, a blood sample is collected from thefundus of the eye, the reactivity of the serum with the antigen istested, for example, by enzyme immunoassay [Antibodies-A LaboratoryManual, Cold Spring Harbor Laboratory (1988)] or the like. An animalshowing a sufficient antibody titer in their sera against the antigenused for the immunization is used as the supply source ofantibody-producing cells for fusion.

Three to seven days after final administration of the antigen, tissuecontaining the antibody-producing cells such as the spleen from theimmunized animal is excised to collect the antibody-producing cells.When the spleen cells are used, the spleen is cut out and loosened,followed by centrifuged. Then, antibody-producing cells for fusion areobtained by removing erythrocytes.

(3) Preparation of Myeloma Cell

An established cell line obtained from mouse is used as myeloma cells.Examples include 8-azaguanine-resistant mouse (derived from BALB/c)myeloma cell line P3-X63Ag8-U1 (P3-U1) [Current Topics in Microbiologyand Immunology, 18, 1 (1978)], P3-NS1/1-Ag41 (NS-1) [European J.Immunology, 6, 511 (1976)], SP2/0-Ag14 (SP-2) [Nature, 276, 269 (1978)],P3-X63-Ag8653 (653) [J. Immunology, 123, 1548 (1979)], P3-X63-Ag8 (X63)[Nature, 256, 495 (1975)] and the like.

The myeloma cells are subcultured in a normal medium [a medium in whichglutamine, 2-mercaptoethanol, gentamicin, FBS and 8-azaguanine are addedto RPMI-1640 medium] and they are subcultured in the normal medium 3 or4 days before cell fusion to ensure the cell number of 2×10⁷ or more onthe day for fusion.

(4) Cell Fusion and Preparation of Hybridoma for Producing MonoclonalAntibody

The antibody-producing cells for fusion obtained by the above (2) andmyeloma cells obtained by the above (3) were sufficiently washed with aminimum essential medium (MEM) or PBS (1.83 g of disodium hydrogenphosphate, 0.21 g of potassium dihydrogen phosphate, 7.65 g of sodiumchloride, 1 liter of distilled water, pH 7.2) and mixed to give a ratioof the antibody-producing cells: the myeloma cells=5 to 10:1, followedby centrifugation. Then, the supernatant is discarded. The precipitatedcell group is sufficiently loosened. After loosening the precipitatedcell, the mixture of polyethylene glycol-1000 (PEG-1000), MEM anddimethylsulfoxide is added to the cell under stirring at 37° C. Inaddition, 1 to 2 mL of MEM medium is added several times every one ortwo minutes, and MEM is added to give a total amount of 50 mL. Aftercentrifugation, the supernatant is discarded. After the cells are gentlyloosen, the cells are gently suspended in HAT medium [a medium in whichhypoxanthine, thymidine and aminopterin is added to the normal medium].The suspension is cultured in a 5% CO₂ incubator for 7 to 14 days at 37°C.

After the culturing, a portion of the culture supernatant is sampled anda hybridoma which is reactive to an antigen containing ASCT2 and is notreactive to an antigen not containing ASCT2 is selected by binding assayas described below. Then, cloning is carried out twice by a limitingdilution method [Firstly, HT medium (HAT medium from which aminopterinis removed) is used, and secondly, the normal medium is used], and ahybridoma which shows a stably high antibody titer is selected as themonoclonal antibody-producing hybridoma.

(5) Preparation of Purified Monoclonal Antibody

The hybridoma cells producing a monoclonal antibody obtained by theabove (4) are administered by intraperitoneal injection into 8- to10-week-old mice or nude mice treated with 0.5 mL of pristane(2,6,10,14-tetramethylpentadecane (pristane) is intraperitoneallyadministered, followed by feeding for 2 weeks). The hybridoma developsascites tumor in 10 to 21 days. The ascitic fluid is collected from themice, centrifuged to remove solids, subjected to salting out with 40 to50% ammonium sulfate and then precipitated by caprylic acid, passedthrough a DEAE-Sepharose column, a protein A column or a gel filtrationcolumn to collect an IgG or IgM fraction as a purified monoclonalantibody.

Furthermore, a monoclonal antibody-producing hybridoma obtained by theabove (4) is cultured in RPMI1640 medium containing 10% FBS or the likeand the supernatant is removed by centrifugation. The precipitated cellsare suspended in Hybridoma SFM medium containing 5% DIGO GF21 andcultured for 3 to 7 days. The purified monoclonal antibody can beobtained by centrifusing the obtained cell suspension, followed bypurifying the resulting supernatant with Protein A colomun or Protein Gcolomn to collect the IgG fractions.

The subclass of the antibody can be determined using a subclass typingkit by enzyme immunoassay. The amount of the protein can be determinedby the Lowry method or from the absorbance at 280 nm.

(6) Selection of Monoclonal Antibody

Selection of monoclonal antibody is carried out by the following bindingassay using an enzyme immunoassay method and inhibition assay ofintracellular uptake of amino acids.

(6-a) Binding Assay

As the antigen, a gene-introduced cell or a recombinant protein obtainedby introducing an expression vector containing a cDNA encoding ASCT2obtained in (1) into Escherichia coli, yeast, an insect cell, an animalcell or the like, or a purified polypeptide or partial peptide obtainedfrom a human tissue is used. When the antigen is a partial peptide, aconjugate is prepared with a carrier protein such as BSA or KLH and isused.

After making these antigens into a solid layer by dispensing in a96-well plate, a substance to be tested such as serum, a culturesupernatant of a hybridoma or a purified monoclonal antibody isdispensed therein as the primary antibody and allowed to react. Afterthoroughly washing with PBS, PBS-Tween, an anti-immunoglobulin antibodylabeled with biotin, an enzyme, a chemiluminescent material, a radiationcompound or the like is dispensed therein as the secondary antibody andallowed to react. After thoroughly washing with PBS-Tween, the reactionis carried out in response to the label of the secondary antibody toselect a monoclonal antibody which specifically reacts with the antigen.

(6-b) Inhibition Assay of Intracellular Uptake of Amino Acids

As an assay cell, a gene-introduced cell in which the expression vectorcomprising cDNA encoding ASCT2 is introduced into a cell such as ananimal cell obtained in the above (1), or a normal or cancer cellexpressing ASCT2 can be used.

The activity of the monoclonal antibody or antibody fragment thereof ofthe present invention to inhibit intracellular uptake of amino acidsthrough ASCT2 is evaluated using a method comprising reacting amonoclonal antibody or an antibody fragment thereof with a normal orcancer cell expressing ASCT2, and then evaluating the inhibition ofglutamine-dependent proliferation using a viable cell counting reagent,or the like [J. Surgical Research, 90, 149 (2000)], a method comprisingreacting a monoclonal antibody or an antibody fragment thereof with anormal or cancer cell expressing ASCT2 and then evaluating theinhibition of uptake of amino acids such as radioactive material-labeledalanine using appropriate equipment such as scintillation counter [J.Biol. Chem., 271, 14883 (1996)] or the like.

2. Preparation of Recombinant Antibody

As production examples of recombinant antibodies, processes forproducing a human chimeric antibody and a humanized antibody are shownbelow.

(1) Construction of Vector for Expression of Recombinant Antibody

A vector for expression of recombinant antibody is an expression vectorfor animal cell into which DNAs encoding CH and CL of a human antibodyhave been inserted, and is constructed by cloning each of DNAs encodingCH and CL of a human antibody into an expression vector for animal cell.

The C region of a human antibody may be CH and CL of any human antibody.Examples include CH belonging to γ1 subclass, CL belonging to x class,and the like. As the DNAs encoding CH and CL of a human antibody, thecDNA may be generally used and a chromosomal DNA comprising an exon andan intron can be also used. As the expression vector for animal cell,any expression vector can be used, so long as a gene encoding the Cregion of a human antibody can be inserted thereinto and expressedtherein. Examples include pAGE107 [Cytotechnol., 3, 133 (1990)], pAGE103[J. Biochem., 101, 1307 (1987)], pHSG274 [Gene, 27, 223 (1984)], pKCR[Proc. Natl. Acad Sci. USA, 78, 1527 (1981)], pSG1bd2-4 [Cytotechnol.,4, 173 (1990)], pSE1UK1Sed1-3 [Cytotechnol., 13, 79 (1993)] and thelike. Examples of a promoter and enhancer used for an expression vectorfor animal cell include an SV40 early promoter [J. Biochem., 101, 1307(1987)], a Moloney mouse leukemia virus LTR [Biochem. Biophys. Res.Commun., 149, 960 (1987)], an immunoglobulin H chain promoter [Cell, 41,479 (1985)] and enhancer [Cell, 33, 717 (1983)] and the like.

The vector for expression of recombinant antibody may be either of atype in which a gene encoding an antibody H chain and a gene encoding anantibody L chain exist on separate vectors or of a type in which bothgenes exist on the same vector (tandem type). In respect of easiness ofconstruction of a vector for expression of recombinant antibody,easiness of introduction into animal cells, and balance between theexpression amounts of antibody H and L chains in animal cells, a tandemtype of the vector for expression of recombinant antibody is morepreferred [J. Immunol. Methods, 167, 271 (1994)]. Examples of the tandemtype of the vector for expression of recombinant antibody includepKANTEX93 (WO 97/10354), pEE18 [Hybridoma, 17, 559 (1998)], and thelike.

(2) Obtaining of cDNA Encoding V Region of Antibody Derived fromNon-Human Animal and Analysis of Amino Acid Sequence

cDNAs encoding VH and VL of non-human anyibody and analysis of aminoacid seaquence are obtained as follows.

mRNA is extracted from hybridoma cells producing an antibody derivedfrom a non-human animal to synthesize cDNA. The synthesized cDNA iscloned into a vector such as a phage or a plasmid, to prepare a cDNAlibrary. Each of a recombinant phage or recombinant plasmid containingcDNA encoding VH or VL is isolated from the library using DNA encoding apart of the C region or V region of an mouse antibody as the probe. Thefull length of the nucleotide sequences of VH and VL of mouse antibodyof interest on the recombinant phage or recombinant plasmid aredetermined, and the full length of the amino acid sequences of VH and VLare deduced from the nucleotide sequences, respectively.

Examples of the non-human animal for preparing a hybridoma cell whichproduces a non-human antibody include mouse, rat, hamster, rabbit, orthe like. Any animals can be used so long as a hybridoma cell can beproduced therefrom.

Examples of the method for preparing total RNA from a hybridoma cellinclude a guanidine thiocyanate-cesium trifluoroacetate method [Methodsin Enzymol., 154, 3 (1987)] using a kit such as RNA easy kit(manufactured by Qiagen) and the like.

Examples of the method for preparing mRNA from total RNA include anoligo (dT) immobilized cellulose column method [Molecular Cloning, ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press(1989)], a method using a kit such as Oligo-dT30<Super>mRNA PurificationKit (manufactured by Takara Bio) and the like. Also, examples of a kitfor preparing mRNA from a hybridoma cell include Fast Track mRNAIsolation Kit (manufactured by Invitrogen), Quick Prep mRNA PurificationKit (manufactured by Pharmacia) and the like.

Examples of the method for synthesizing cDNA and preparing a cDNAlibrary include known methods [Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Lab. Press (1989); Current Protocols in MolecularBiology, Supplement 1, John Wiley & Sons (1987-1997)]; a method using akit such as Super Script Plasmid System for cDNA Synthesis and PlasmidCloning (manufactured by GIBCO BRL), ZAP-cDNA Kit (manufactured byStratagene), etc.; and the like.

The vector into which the synthesized cDNA using mRNA extracted from ahybridoma cell as the template is inserted for preparing a cDNA librarymay be any vector, so long as the cDNA can be inserted. Examples includeZAP Express [Strategies, 5, 58 (1992)], pBluescript II SK(+) [NucleicAcids Research, 17, 9494 (1989)], kzapII (manufactured by Stratagene),λgt10 and λgt11 [DNA Cloning: A Practical Approach, I, 49 (1985)],Lambda BlueMid (manufactured by Clontech), λExCell and pT7T3 18U(manufactured by Pharmacia), pcD2 [Mol. Cell. Biol., 3, 280 (1983)],pUC18 [Gene, 33, 103 (1985)], and the like.

Any Escherichia coli for introducing the cDNA library constructed by aphage or plasmid vector may be used, so long as the cDNA library can beintroduced, expressed and maintained. Examples include KL1-Blue MRF′[Strategies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088 andY1090 [Science, 222: 778 (1983)], NM522 [J. Mol. Biol., 166, 1 (1983)],K802 [J. Mol. Biol., 16, 118 (1966)], JM105 [Gene, 38, 275 (1985)], andthe like.

A colony hybridization or plaque hybridization method using an isotope-or fluorescence-labeled probe may be used for selecting cDNA clonesencoding VH and VL of a non-human antibody or the like 1 from the cDNAlibrary [Molecular Cloning, A Laboratory Manual, Second Edition, ColdSpring Harbor Laboratory Press (1989)]. Also, the cDNAs encoding VH andVL can be prepared through polymerase chain reaction (hereinafterreferred to as “PCR”; Molecular Cloning, A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press (1989); Current Protocolsin Molecular Biology, Supplement 1, John Wiley & Sons (1987-1997)) bypreparing primers and using cDNA prepared from mRNA or a cDNA library asthe template.

The nucleotide sequence of the cDNA can be determined by digesting thecDNA selected with appropriate restriction enzymes and the like, cloningthe fragments into a plasmid such as pBluescript SK(−) (manufactured byStratagene), carrying out the reaction by a usually used nucleotideanalyzing method such as analyzing the sequence using an automaticnucleotide sequence analyzer such as A.L.F. DNA sequencer (manufacturedby Pharmacia) after the dideoxy method [gProc. Natl. Acad. Sci. USA, 74,5463 (1977)].

Whether the obtained cDNAs encode the full amino acid sequences of VLand VL of the antibody containing a secretory signal sequence can beconfirmed by estimating the full length of the amino acid sequences ofVH and VL from the determined nucleotide sequence and comparing themwith the full length of the amino acid sequences of VH and VL of knownantibodies [Sequences of Proteins of Immunological Interest, US Dept.Health and Human Services (1991)]. The length of the secretory signalsequence and N-terminal amino acid sequence can be deduced by comparingthe full length of the amino acid sequences of VH and VL of the antibodycomprising a secretory signal sequence with full length of the aminoacid sequences of VH and VL of known antibodies [Sequences of Proteinsof Immunological Interest, US Dept. Health and Human Services (1991)],and the subgroup to which they belong can also be known. Furthermore,the amino acid sequence of each of CDRs of VH and VL can be found bycomparing the obtained amino acid sequences with amino acid sequences ofVH and VL of known antibodies [Sequences of Proteins of ImmunologicalInterest, US Dept. Health and Human Services (1991)].

Moreover, the novelty of the full length of the amino acid sequence ofVH and VL can be examined by carrying out a homology search withsequences in any database, for example, SWISS-PROT, PIR-Protein or thelike using the obtained full length of the amino acid sequences of VHand VL, for example, according to the BLAST method [J. Mol. Biol., 215,403 (1990)] or the like.

(3) Construction of Vector for Expression of Human Chimeric Antibody

cDNAs encoding VH and \TL of antibody of non-human animal arerespectively cloned in the upstream of genes encoding CH or CL of humanantibody of vector for expression of recombinant antibody mentioned inthe above (1) to thereby construct a vector for expression of humanchimeric antibody. For example, in order to ligate cDNA comprising anucleotide sequence of 3′-terminal of VH or VL of antibody of non-humananimal and a nucleotide sequence of 5′-terminal of CH or CL of humanantibody, each cDNA encoding VH and VL of antibody of non-human animalis prepared so as to encodes approptiate amino acids encoded by anucleotide sequence of a linkage portion and designed to have anappropriate recognition sequence of a restriction enzyme. The obtainedcDNAs encoding VH and VL of antibody are respectively cloned so thateach of them is expressed in an appropriate form in the upstream of geneencoding CH or CL of human antibody of the vector for expression ofhumanized antibody mentioned in the above (1) to construct a vector forexpression of human chimeric antibody.

In addition, cDNA encoding VH or VL of non-human animal is amplified byPCR using a synthetic DNA having a recognition sequence of anappropriate restriction enzyme at both ends and each of them is clonedto the vector for expression of recombinant antibody obtained in theabove (1).

(4) Construction of cDNA Encoding V Region of Humanized Antibody

cDNAs encoding VH or VL of a humanized antibody can be obtained asfollows. First, amino acid sequences of framework region (hereinafterreferred to as “FR”) in VH or VL of a human antibody to which amino acidsequences of CDRs in VH or VL of an antibody derived from a non-humananimal antibody are transplanted are respectively selected. Any aminoacid sequences of FR of a human antibody can be used, so long as theyare derived from human. Examples include amino acid sequences of FRs ofhuman antibodies registered in database such as Protein Data Bank or thelike, and amino acid sequences common to subgroups of FRs of humanantibodies [Sequences of Proteins of Immunological Interest, US Dept.Health and Human Services (1991)], and the like. In order to inhibit thedecrease in the binding activity of the antibody, amino acid sequenceshaving high homology (at least 60% or more) with the amino acid sequenceof FR in VH or VL of the original antibody is selected.

Then, amino acid sequences of CDRs of the original antibody are graftedto the selected amino acid sequence of FR in VH or VL of the humanantibody, respectively, to design each amino acid sequence of VH or VLof a humanized antibody. The designed amino acid sequences are convertedto DNA sequences by considering the frequency of codon usage found innucleotide sequences of genes of antibodies [Sequence of Proteins ofImmunological Interest, US Dept. Health and Human Services (1991)], andthe DNA sequence encoding the amino acid sequence of VH or VL of ahumanized antibody is designed.

Based on the designed nucleotide sequences, several synthetic DNAshaving a length of about 100 nucleotides are synthesized, and PCR iscarried out using them. In this case, it is preferred that 6 syntheticDNAs per each of the H chain and the L chain are designed in view of thereaction efficiency of PCR and the lengths of DNAs which can besynthesized.

Furthermore, the cDNA encoding VH or VL of a humanized antibody can beeasily cloned into the vector for expression of humanized antibodyconstructed in (1) by introducing the recognition sequence of anappropriate restriction enzyme to the 5′ terminal of the synthetic DNAsexisting on the both ends.

After the PCR, an amplified product is cloned into a plasmid such aspBluescript SK (−) (manufactured by Stratagene) or the like, and thenucleotide sequence is determined according to a method similar to themethod described in (2) to obtain a plasmid having a DNA sequenceencoding the amino acid sequence of VH or VL of a desired humanizedantibody.

(5) Modification of Amino Acid Sequence of V Region of HumanizedAntibody

It is known that when a humanized antibody is produced by simplygrafting only CDRs in VH and VL of an antibody derived from a non-humananimal into FRs of VH and VL of a human antibody, its antigen bindingactivity is lower than that of the original antibody derived from anon-human animal [BIO/TECHNOLOGY, 9,266 (1991)]. In humanizedantibodies, among the amino acid sequences of FRs in VH and VL of ahuman antibody, an amino acid residue which directly relates to bindingto an antigen, an amino acid residue which interacts with an amino acidresidue in CDR, and an amino acid residue which maintains thethree-dimensional structure of an antibody and indirectly relates tobinding to an antigen is identified and modified to an amino acidresidue which is found in the original non-humahumanized antibody tothereby increase the antigen binding activity which has been decreased.

In order to identify the amino acid residues relating to the antigenbinding activity in FR, the three-dimensional structure of an antibodyis constructed and analyzed by X-ray crystallography [J. Mol. Biol.,112, 535 (1977)], computer-modeling [Protein Engineering, 7, 1501(1994)] or the like. In addition, various attempts must be currently benecessary, for example, several modified antibodies of each antibody areproduced and the correlation between each of the modified antibodies andits antibody binding activity is examined.

The modification of the amino acid sequence of FR in VH and VL of ahuman antibody can be accomplished using various synthetic DNA formodification according to PCR as described in (4). With regard to theamplified product obtained by the PCR, the nucleotide sequence isdetermined according to the method as described in (2) so that whetherthe objective modification has been carried out is confirmed.

(6) Construction of Vector for Expression of Humanized Antibody

A vector for expression of humanized antibody can be constructed bycloning each cDNA encoding VH or VL of a constructed recombinantantibody into upstream of each gene encoding CH or CL of the humanantibody in the vector for expression of recombinant antibody asdescribed in (1).

For example, when recognizing sequences of an appropriate restrictionenzymes are introduced to the 5′-terminal of synthetic DNAs positionedat both ends among synthetic DNAs used in the construction of VH or VLof the humanized antibody in (4) and (5), cloning can be carried out sothat they are expressed in an appropriate form in the upstream of eachgene encoding CH or CL of the human antibody in the vector forexpression of humanized antibody as described in the above (1).

(7) Transient Expression of Recombinant Antibody

In order to efficiently evaluate the antigen binding activity of varioushumanized antibodies produced, the recombinant antibodies can beexpressed transiently using the vector for expression of recombinantantibody as described in (3) and (6) or the modified expression vectorthereof.

Any cell can be used as a host cell, so long as the host cell canexpress a recombinant antibody. Generally, COS-7 cell (ATCC CRL1651) isused [Methods in Nucleic Acids Res., CRC Press, 283 (1991)]. Examples ofthe method for introducing the expression vector into COS-7 cell includea DEAE-dextran method [Methods in Nucleic Acids Res., CRC Press, 283(1991)], a lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413(1987)], and the like.

After introduction of the expression vector, the expression amount andantigen binding activity of the recombinant antibody in the culturesupernatant can be determined by the enzyme immunoassay [MonoclonalAntibodies Principles and practice, Third edition, Academic Press(1996), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory(1988), Monoclonal Antibody Experiment Manual, Kodansha Scientific(1987)] and the like.

(8) Obtaining Transformant which Stably Expresses Recombinant Antibodyand Preparation of Recombinant Antibody

A transformant which stably expresses a recombinant antibody can beobtained by introducing the vector for expression of recombinantantibody described in (3) and (6) into an appropriate host cell.

Examples of the method for introducing the expression vector into a hostcell include electroporation [Japanese Published Unexamined PatentApplication No. 257891/90, Cytotechnology, 3, 133 (1990)] and the like.

As the host cell into which a vector for expression of a recombinantantibody is introduced, any cell can be used, so long as it is a hostcell which can produce the recombinant antibody. Examples include mouseSP2/0-Ag14 cell (ATCC CRL1581), mouse P3×63-Ag8.653 cell (ATCC CRL1580),CHO cell in which a dihydrofolate reductase gene (hereinafter referredto as “dhfr”) is defective [Proc. Natl. Acad. Sci. U.S.A., 77, 4216(1980)], lection resistance-acquired Lec13 [Somatic Cell and Moleculargenetics, 12, 55 (1986)], CHO cell in which α1,6-fucosyltransaferse geneis defected (WO 2005/35586, WO 02/31140), rat YB2/3HL.P2.G11.16Ag.20cell (ATCC CRL1662), and the like.

In addition, host cells in which activity of a protein such as an enzymerelating to synthesis of an intracellular sugar nucleotide, GDP-fucose,a protein such as an enzyme relating to the modification of a sugarchain in which 1-position of fucose is bound to 6-position ofN-acetylglucosamine in the reducing end through α-bond in a complex typeN-glycoside-linked sugar chain, or a protein relating to transport of anintracellular sugar nucleotide, GDP-fucose, to the Golgi body areintroduced is decreased or deleted, preferably CHO cell in whichα1,6-fucosyltransferase gene is defected as described in WO05/35586,WO02/31140 or the like, can also be used.

After introduction of the expression vector, transformants which expressa recombinant antibody stably are selected by culturing in a medium foranimal cell culture containing an agent such as G418 sulfate(hereinafter referred to as “G418”) or the like (Japanese PublishedUnexamined Patent Application No. 257891/90).

Examples of the medium for animal cell culture include RPM:11640 medium(manufactured by Invitrogen), GIT medium (manufactured by NihonPharmaceutical), EX-CELL301 medium (manufactured by JRH), IMDM medium(manufactured by Invitrogen), Hybridoma-SFM medium (manufactured byInvitrogen), media obtained by adding various additives such as fetalcalf serum (hereinafter referred to as “FCS”) to these media, and thelike. The recombinant antibody can be produced and accumulated in aculture supernatant by culturing the selected transformants in a medium.The expression amount and antigen binding activity of the recombinantantibody in the culture supernatant can be measured by ELISA or thelike. Also, in the transformant, the expression amount of therecombinant antibody can be increased by using DHFR amplification systemor the like according to the method disclosed in Japanese PublishedUnexamined Patent Application No. 257891/90.

The recombinant antibody can be purified from the culture supernatant ofthe transformant by using a protein A column [MonoclonalAntibodies-Principles and practice, Third edition, Academic Press(1996), Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory(1988)]. For example, the recombinant antibody can be purified by acombination of gel filtration, ion-exchange chromatography,ultrafiltration and the like. The molecular weight of the H chain or theL chain of the purified recombinant antibody or the antibody molecule asa whole is determined by polyacrylamide gel electrophoresis (hereinafterreferred to as “SDS-PAGE”) [Nature, 227, 680 (1970)], Western blotting[Monoclonal Antibodies-Principles and practice, Third edition, AcademicPress (1996), Antibodies-A Laboratory Manual, Cold Spring HarborLaboratory (1988)], and the like.

3. Activity Evaluation of the Monoclonal Antibody or Antibody Fragment

The activity of the purified monoclonal antibody or antibody fragment ofthe present invention can be evaluated in the following manner.

The binding activity to ASCT2-expressing cell is evaluated by thebinding assay described in the above 1 (6a). Furthermore, it can bemeasured by fluorescent antibody technique [Cancer Immunol. Immunother.,36, 373 (1993)], a surface plasmon resonance method using such asBIAcore system or the like.

The inhibitory activity on intracellular uptake of amino acids by ASCT2can be evaluated by the method described in the above 1-(6b).

In addition, CDC activity or ADCC activity against an antigen positivecell line is evaluated by a known method [Cancer Immunol. Immunother.,36, 373 (1993)].

4. Method for Treating Disease Using the Anti-ASCT2 Monoclonal Antibodyor Antibody Fragment of the Present Invention

A monoclonal antibody which specifically recognizes a nativethree-dimensional structure of ASCT2 and binds to the extracellularregion, or an antibody fragment thereof, of the present invention can beused for treating a disease relating to ASCT2.

The therapeutic agent comprising the monoclonal antibody or antibodyfragment of the present invention or derivatives thereof may be only theantibody or antibody fragment or derivatives thereof as an activeingredient, and is preferably supplied as a pharmaceutical preparationproduced by an appropriate method well known in the technical field ofpharmaceutics, by mixing it with one or more pharmaceutically acceptablecarriers.

Examples of a route of administration include oral administration andparenteral administration, such as buccal, tracheal, rectal,subcutaneous, intramuscular or intravenous administration. Examples ofthe dosage form includes sprays, capsules, tablets, powder, granules,syrups, emulsions, suppositories, injections, ointments, tapes and thelike.

The pharmaceutical preparation suitable for oral administration includesemulsions, syrups, capsules, tablets, powders, granules and the like.

Liquid preparations such as emulsions and syrups can be produced using,as additives, water; sugars such as sucrose, sorbitol and fructose;glycols such as polyethylene glycol and propylene glycol; oils such assesame oil, olive oil and soybean oil; antiseptics such asp-hydroxybenzoic acid esters; flavors such as strawberry flavor andpeppermint; and the like.

Capsules, tablets, powders, granules and the like can be produced using,as additives, excipients such as lactose, glucose, sucrose and mannitol;disintegrating agents such as starch and sodium alginate; lubricantssuch as magnesium stearate and talc; binders such as polyvinyl alcohol,hydroxypropylcellulose and gelatin; surfactants such as fatty acidester; plasticizers such as glycerin; and the like.

The pharmaceutical preparation suitable for parenteral administrationincludes injections, suppositories, sprays and the like.

Injections can be prepared using a carrier such as a salt solution, aglucose solution or a mixture of both thereof.

Suppositories can be prepared using a carrier such as cacao butter,hydrogenated fat or carboxylic acid.

Sprays can be prepared using the antibody or antibody fragment as suchor using it together with a carrier which does not stimulate the buccalor airway mucous membrane of the patient and can facilitate absorptionof the compound by dispersing it as fine particles. The carrier includeslactose, glycerol and the like. It is possible to produce pharmaceuticalpreparations such as aerosols and dry powders.

In addition, the components exemplified as additives for oralpreparations can also be added to the parenteral preparations.

5. Method for Diagonosing Disease Using the Anti-ASCT2 MonoclonalAntibody or Antibody Fragment of the Present Invention

A disease relating to ASCT2 can be diagnosed by detecting or determiningASCT2 or a cell expressing ASCT2 using the monoclonal antibody orantibody fragment of the present invention.

A diagonosis of cancer, one of the diseases relating to ASCT2, can becarried out by, for example, the detection or measurement of ASCT2 asfollows.

Firstly, on the living body samples collected from two or more of theliving bodies of healthy persons, the expressed amount of ASCT2 in theliving body samples of healthy persons is confirmed by carrying outdetection or measurement of ASCT2 by the following immunological meansusing the monoclonal antibody or antibody fragment of the presentinvention or derivatives thereof. By examining the amount of ASCT2 alsoin the living body samples of the person to be tested in the samemanner, the amount is compared with the amount in healthy persons. Whenthe amount of the polypeptide in the person to be tested is increased incomparison with the healthy persons, it can be diagnosed that cancer ispositive.

An immunological method is a method in which an antibody amount or anantigen amount is detected or determined using a labeled antigen orantibody.

Examples of the immunological method include radioactivesubstance-labeled immunoantibody method, enzyme immunoassay, fluorescentimmunoassay, luminescent immunoassay, Western blotting method,physico-chemical means and the like.

Examples of the radioactive substance-labeled immunoantibody methodinclude a method, in which the antibody or antibody fragment of thepresent invention is allowed to react with an antigen or a cellexpressing an antigen, then anti-immunoglobulin antibody subjected toradioactive labeling, a binding fragment thereof or the like is allowedto react therewith, followed by determination using a scintillationcounter or the like.

Examples of the enzyme immunoassay include a method, in which theantibody or antibody fragment of the present invention is allowed toreact with an antigen or a cell expressing an antigen or the like, thenan anti-immunoglobulin antibody or an binding fragment thereof subjectedto antibody labeling is allowed to react therewith and the coloredpigment is measured by a spectrophotometer, and, for example, sandwichELISA may be used. As a label used in the enzyme immunoassay, any knownenzyme label [Enzyme Immunoassay, published by Igaku Shoin (1987)] canbe used. Examples include alkaline phosphatase labeling, peroxidaselabeling, luciferase labeling, biotin labeling and the like.

Sandwich ELISA is a method in which an antibody is bound to a solidphase, antigen to be detected or measured is trapped and anotherantibody is allowed to react with the trapped antigen. In the ELISA, twokinds of antibody which recognizes the antigen to be detected ormeasured or the antibody fragment thereof in which antigen recognizingsite is different are prepared and one antibody or antibody fragments ispreviously adsorbed on a plate (such as a 96-well plate) and anotherantibody or antibody fragment is labeled with a fluorescent substancesuch as FITC, an enzyme such as peroxidase, or biotin. The plate towhich the above antibody is adsorbed is allowed to react with the cellseparated from living body or disrupted cell suspension thereof, tissueor disintegrated solution thereof, cultured cells, serum, pleuraleffusion, ascites, eye solution or the like, then allowed to react withlabeled monoclonal antibody or antibody fragment and a detectionreaction corresponding to the labeled substance is carried out. When anantigen concentration in the sample to be tested is measured by themethod, antigen concentration in the sample to be tested can becalculated from a calibration curve prepared by a stepwise dilution ofantigen of known concentration. As an antibody used for sandwich ELISA,any of a polyclonal antibody and a monoclonal antibody may be used orantibody fragments such as Fab, Fab′ and F(ab)₂ may be used. As acombination of two kinds of antibodies used in sandwich ELISA, acombination of monoclonal antibodies or antibody fragments recognizingdifferent epitopes may be used or a combination of polyclonal antibodywith monoclonal antibody or antibody fragments may be used.

A fluorescent immunoassay includes a method described in the literatures[Monoclonal Antibodies—Principles and practice, Third Edition, AcademicPress (1996); Manual for Monoclonal Antibody Experiments, KodanshaScientific (1987)] and the like. As a label for the fluorescentimmunoassay, any of known fluorescent labels (Fluorescent Immunoassay,Soft Science, (1983)) may be used as described already. Examples includeFITC, RITC and the like.

The luminescent immunoassay can be carried out using the methodsdescribed in the literature [Bioluminescence and Chemical Luminescence,Rinsho Kensa, 42, Hirokawa Shoten (1998)] and the like. As a label usedfor luminescent immunoassay, any of known luminescent labels can beincluded. Examples include acridinium ester, lophine or the like may beused.

Western blotting is a method in which an antigen or a cell expressing anantigen is fractionated by SDS-polyacrylamide gel electrophoresis[Antibodies-A Laboratory Manual (Cold Spring Harbor Laboratory, 1988)],the gel is blotted onto PVDF membrane or nitrocellulose membrane, themembrane is allowed to react with antigen-recognizing antibody orantibody fragment, further allowed to react with an anti-mouse IgGantibody or antibody fragment which is labeled with a fluorescentsubstance such as FITC, an enzyme label such as peroxidase, a biotinlabeling, or the like, and the label is visualized to confirm thereaction. An example thereof is described below. Cells or tissues inwhich a polypeptide having the amino acid sequence represented by SEQ IDNO:2 is expressed are dissolved in a solution and, under reducingconditions, 0.1 to 30 μg as a protein amount per lane is electrophoresedby an SDS-PAGE method. The electrophoresed protein is transferred to aPVDF membrane and allowed to react with PBS containing 1 to 10% of BSA(hereinafter referred to as “BSA-PBS”) at room temperature for 30minutes for blocking. Here, the monoclonal antibody of the presentinvention is allowed to react therewith, washed with PBS containing 0.05to 0.1% Tween 20 (hereinafter referred to as “Tween-PBS”) and allowed toreact with goat anti-mouse IgG labeled with peroxidase at roomtemperature for 2 hours. It is washed with Tween-PBS and a band to whichthe monoclonal antibody is bound is detected using ECL Western BlottingDetection Reagents (manufactured by Amersham) or the like to therebydetect a polypeptide having the amino acid sequence represented by SEQID NO:2. As an antibody used for the detection in Western blotting, anantibody which can be bound to a polypeptide having no three-dimensionalstructure of a natural type is used.

The physicochemical method is specifically carried out by reacting ASCT2as the antigen with the antibody or antibody fragment of the presentinvention to form an aggregate, and detecting this aggregate. Otherexamples of the physicochemical methods include a capillary method, aone-dimensional immunodiffusion method, an immunoturbidimetry, a lateximmunoturbidimetry [Handbook of Clinical Test Methods, Kanehara Shuppan,(1988)] and the like. For example, in a latex immunodiffusion method, acarrier such as polystyrene latex having a particle size of about of 0.1to 1 μm sensitized with antibody or antigen may be used and when anantigen-antibody reaction is carried out using the corresponding antigenor antibody, scattered light in the reaction solution increases whiletransmitted light decreases. When such a change is detected asabsorbance or integral sphere turbidity, it is now possible to measureantigen concentration, etc. in the sample to be tested.

On the other hand, for the detection or measurement of the cellexpressing ASCT2, known immunological detection methods can be used, andan immunoprecipitation method, a immuno cell staining method, an immunetissue staining method, a fluorescent antibody staining method and thelike are preferably used.

An immunoprecipitation method is a method in which a cell expressingASCT2 is allowed to react with the monoclonal antibody or antibodyfragment of the present invention and then a carrier having specificbinding ability to immunoglobulin such as protein G-Sepharose is addedso that an antigen-antibody complex is precipitated. Also, the followingmethod can be carried out. The monoclonal antibody or antibody fragmentof the present invention is solid-phased on a 96-well plate for ELISAand then blocked with BSA-PBS. When the antibody is in a non-purifiedstate such as a culture supernatant of hybridoma cell, anti-mouseimmunoglobulin or rat immunoglobulin or protein A or G or the like ispreviously adsorbed on a 96-well plate for ELISA and blocked withBSA-PBS and a culture supernatant of hybridoma cell is dispensed theretofor binding. After BSA-PBS is discarded and the residue is sufficientlywashed with PBS, reaction is carried out with a dissolved solution ofcells or tissues expressing ASCT2. An immune precipitate is extractedfrom the well-washed plate with a sample buffer for SDS-PAGE anddetected by the above-described Western blotting.

An immune cell staining method and an immune tissue staining method aremethods where cells or tissues in which antigen is expressed aretreated, if necessary, with a surfactant, methanol or the like to makean antibody easily permeate to the cells or tissues, then the monoclonalantibody of the present invention is allowed to react therewith, thenfurther allowed to react with an anti-immunoglobulin antibody or bindingfragment thereof subjected to fluorescent labeling such as FITC, enzymelabeling such as peroxidase or biotin labeling and the label isvisualized and observed under a microscope Also, the detection can becarried out by a fluorescent antibody staining method [MonoclonalAntibodies—Principles and practice, Third Edition, Academic Press(1996), Manual for Experiments of Monoclonal Antibodies, KodanshaScientific (1987)] in which cells are allowed to react with afluorescence-labeled antibody and analyzed by a flow cytometer.Particularly, since the monoclonal antibody or antibody fragment of thepresent invention binds to an extracellular region of ASCT2, it can bepreferably used for detection of a cell expressing the polypeptidemaintaining a natural type three-dimensional structure by a fluorescentantibody staining method.

In addition, in the case of using FMAT8100HTS system (manufactured byApplied Biosystems) and the like among fluorescent antibody stainingmethods, the antigen quantity or antibody quantity can be measuredwithout separating the formed antibody-antigen complex and the freeantibody or antigen which is not concerned in the formation of theantibody-antigen complex.

The present invention is described below by Examples; however, thepresent invention is not limited to the following Examples.

Example 1 Analysis of ASCT2 Gene Expression in Various Cell Lines,Xenografts, and Normal Tissues

(1) Construction of Xenografts with Subcutaneous Transplantation of aCancer Cell Line into SCID Mice and Preparation of Tumor Mass

In accordance with the following procedure, human cancer cell lines weresubcutaneously transplanted into SCID mice to thereby constructxenografts. A tumor mass was extracted and prepared from the resultingxenografts.

Cells derived from human pancreatic cancer cell lines [ASPC-1 (ATCCAccession No. CRL-1682), CaPan-1 (ATCC Accession No. HTB-79), PANC-1(ATCC Accession No. CRL-1469)], and human colorectal cancer cell lines[Colo205 (Riken (Physicochemical Research Institute) Cell Bank No.RCB2127), HT-29 (ATCC Accession No. HTB-38), LS180 (ATCC Accession No.CCL-187), SW1116 (ATCC Accession No. CCL-233), and WiDr (ATCC AccessionNo. CCL-218)] were suspended in PBS to give a cell density of about1×10⁸ cells/mL. Into the ventral hypodermis of 4 Fox CHASEC.B-17/Icr-scidJcl mice per group (male, 5 weeks old, purchased fromCLEA Japan), 100 μL/animal of each cell suspension was transplanted.

Each of the cells were suspended and subcultured in an RPM 1640 medium(manufactured by Invitrogen) containing 10% inactivated fetal bovineserum (manufactured by Invitrogen), in a CO₂ incubator at 37° C. to usefor transplantation.

The resulting xenografts were named as xASPC1, xCaPanl, xPANC1,xColo205, xHT29, xLS180, xSW1116, and xWiDr, respectively.

After tumor transplantation, the diameter of tumor mass was measured dayby day using vernier calipers. The animals in which the major axis ofthe tumor became about 1 cm were sequentially sacrificed by bleedingunder anesthesia and then each tumor mass was excised. Each tumor masswas cut into 4 portions and quickly frozen using liquid nitrogen,followed by storage in a freezer at −80° C.

(2) Extraction of Total RNA and Purification of Poly A(+) RNA

From cell lines and tumor mass of xenografts prepared in the above (1),total RNA was extracted and poly A(+) RNA was purified in accordancewith the following procedure.

As a cell line, blood cancer-derived cell line {KG-1 (ATCC Accession No.CCL-246), THP-1 (ATCC Accession No.TIB-202), HL-60 (ATCC Accession No.CCL-240) [all, acute myeloid leukemia (AML) derived cell line], CCRF-CEM(ATCC Accession No. CCL-119), CCRF-SB (ATCC Accession No. CCL-120),Jurkat (ATCC Accession No. TIB-152), HSB-2 (ATCC Accession No. CCL-120),HPB-ALL (Riken Cell Bank No.:RCB1935) [all, acute lymphatic leukemia(ALL) derived cell line], K-562 (ATCC Accession No. CCL-243), KU812(ATCC Accession No. CRL-2099) [all, chronic myelocytic leukemia (CML)derived cell line], KMS-11 (HSRRB No. JCRB 1179), ARH-77 (ATCC AccessionNo. CRL-1621), IM-9 (ATCC Accession No. CCL-159), RPMI18226 (HSRRB No.JCRB0034), U266B1 (ATCC Accession No. T1E-196), MC/CAR (ATCC AccessionNo. CRL-8083) [all, multiple myeloma (MM) derived cell line], HS-Sultan(ATCC Accession No. CRL-1484), Daudi (ATCC Accession No. CCL-213), Raji(ATCC Accession No. CCL-86), Ramos (ATCC Accession No. CRL-1596) [all,Burkitt's lymphoma (BL) derived cell line], U-937 (ATCC Accession No.CRL-1593.2), ML-1 (DSMZ No. ACC464) [all, histiocytic lymphoma (HS)derived cell line]}, lung cancer derived cell line [PC-14 (Riken CellBank No.:RCB0446), PC-7 (Immuno-Biological Laboratories Co., Ltd.;Product No.:37011), PC-9 (Immuno-Biological Laboratories Co., Ltd.;Product No. 37012), PC-1 (Immuno-Biological Laboratories Co., Ltd.;Product No. 37008) (all, non-small cell lung cancer), Lu-139 (Riken CellBank No.:RCB0469), NCI-H69 (ATCC Accession No. HTB-119), RERF-LC-MA(HSRRB No. JCR130812), SBC-5 (HSRRB No. JCRB0819) (all, small-cell lungcancer)], gastric cancer derived cell line [Kato III (Riken Cell BankNo. RCB2088), MKN-74 (HSRRB No. JCRB0255), NUGC-4 (Riken Cell Bank No.RCB 1939), AZ-521 (HSRRB No. JCRB0061)], colorectal cancer derived cellline {Colo205 (Riken Cell Bank No. RCB2127), HT-29 (ATCC Accession No.HTB-38), LS174T[European Collection of Cell Cultures (ECACC) No.87060401], LS180 (ATCC Accession No. CCL187), SW1116 (ATCC Accession No.CCL-233)}, pancreatic cancer derived cell line [ASPC-1 (ATCC AccessionNo. CRL-1682), BXPC-3 (ATCC Accession No. CRL-1687), CaPan-1 (ATCCAccession No. HTB-79)], malignant melanoma (melanoma) derived cell line[G-361 (ATCC Accession No. CRL-1424), BMV-1 (Riken Cell Bank No.RCB0004), SK-MEL-28 (ATCC Accession No. HTB-72)], and normal human lungfibroblast [MRC-5 (ATCC Accession No. CCL-171)] were used.

The extraction of total RNA from the cell lines was carried out asfollows. In the case of adhesive cell lines, the medium was removedusing an aspirator after culturing, washing with an appropriate volumeof PBS was carried out and cells were recovered using a spatula made ofsilicone. They were sufficiently suspended and lysed by adding 1 mL of aTRIzol Reagent (manufactured by Invitrogen) per the cells correspondingto 10 cm² of cultured area. In addition, the resulting cell lysate waspassed through an 18-gauge injection needle 10 times to cleave thegenomic DNA into pieces. In the case of floating cell lines, the cellculture was centrifuged at 1,500 rpm for 5 minutes using a refrigeratedcentrifuge (manufactured by Hitachi Koki, Himac CF15R, rotor: T11A21),the medium was removed by decantation and the cells were suspended inPBS. The cell suspension was centrifuged again at 1,500 rpm for 5minutes using a refrigerated centrifuge (manufactured by Hitachi Koki,Himac CF15R, rotor: T11A21) and the cells were recovered. To 1×10⁷recovered cells, 1 mL of a TRIzol Reagent (manufactured by Invitrogen)was added and suspended sufficiently so as to lyse cells. The celllysate was passed through an 18-gauge injection needle 10 times tocleave the genomic DNA into pieces.

Lysis of tumor mass of the xenografts prepared in (1) and extraction oftotal RNA were carried out as follows. Frozen tumor mass was poured into10 mL of a TRIzol Reagent (manufactured by Invitrogen) and immediatelylysed using a Polytron homogenizer (PT 2100, manufactured by Kinematica)at 30,000 rpm for 15 seconds to give cell lysates. Each of the celllysates was centrifuged at 11,000 rpm for 10 minutes using arefrigerated centrifuge (manufactured by Hitachi Koki, Himac CF15R,rotor: T11A21) and each of the supernatants was transferred to a freshtube carefully so as not to carry over the precipitate. To thesupernatant, 2 mL of chloroform was added and vigorously stirred for 15seconds and the mixture was allowed to stand at room temperature for 2to 3 minutes and centrifuged at 3,000 rpm for 90 minutes at 4° C. usinga refrigerated centrifuge (manufactured by Hitachi Koki, Himac CF7D2,rotor: RT3S3). Each of the supernatants was transferred to a fresh tube,and 5 mL of isopropanol was added thereto, followed by gentle mixing.The mixture was allowed to stand at room temperature for 10 minutes andcentrifuged at 11,000 rpm for 10 minutes using a refrigerated centrifuge(manufactured by Hitachi Koki, Himac CF15R, rotor: T11A21) and, afterremoving the supernatant, 10 mL of a 75% aqueous ethanol solution wasadded thereto, followed by mixing and centrifugation at 11,000 rpm for 5minutes using a refrigerated centrifuge (manufactured by Hitachi Koki,Himac CF15R, rotor: T11A21) to give a precipitate. The precipitate wasdissolved in an appropriate volume of RNase-free water to prepare atotal RNA sample. The concentration of the total RNA sample was measuredby an absorption spectrophotometer and it was confirmed that the ratioof A260/A280 was 1.7 or more. When the ratio of A260/A280 was less than1.7, further purification was carried out using an RNeasy kit(manufactured by Qiagen).

From 400 μg of the total RNA sample prepared as above, poly A(+) RNA waspurified using a Micro Poly(A) Pure Kit (manufactured by Ambion) inaccordance with the instructions attached thereto.

(3) Synthesis of cDNA

cDNA was synthesized from the poly A(+) RNA obtained in (2) orcommercially available tissue-derived mRNA using a SuperScriptFirst-Strand Synthesis System for RT-PCR (manufactured by Invitrogen).

As mRNA derived from human normal tissues (liver, lung, whole brain,heart, stomach, spleen, spinal cord, small intestine, skeletal muscle,uterus, respiratory tract, thyroid gland, thymus gland, testis, salivarygland, prostate gland, placenta, lymph node, pancreas, large intestine,blood, or kidney), commercially available mRNA (manufactured byClontech) was used. As mRNA derived from the human clinical cancertissues (lung cancer, gastric cancer, colorectal cancer, kidney cancer,liver cancer, uterine cancer, breast cancer, or esophageal cancer),commercially available RNA (manufactured by BioChain) was also used.

To 1 μg of poly A(+) RNA obtained in (2) or commercially availabletissue-derived mRNA, 1 μL of 10 mmol/L dNTPs and 1 μL of a 0.5 μg/μLOligo (dT)₁₂₋₁₈ were added, and then DEPC water was added thereto togive a total volume of 7 μL. The reaction solution was heated todenaturation at 65° C. for 5 minutes, quenched on ice and allowed tostand for 1 minute or longer. To the mRNA solution, 10×RT buffer (2 μL),magnesium chloride (4 μL, 25 mmol/L), 0.1 mol/L DTT (2 μL) and RNase OUTRecombinant Ribonuclease Inhibitor (1 μL) were added and the temperaturewas kept at 42° C. for 2 minutes. SuperscriptII RT (14) was furtheradded thereto to carry out reverse transcription reaction at 42° C. for50 minutes. The enzyme was inactivated by heating at 70° C. for 15minutes. Then, 1 μL of E. coli RNase H was added to reaction solutionand reacted at 37° C. for 20 minutes. DEPC water was added to theresulting solution to give a total volume of 1 mL.

Hereinafter, the reaction system of real-time PCR employed 10 μL of afive-fold dilution of the above-prepared solution.

(4) Quantitative Determination of Expression Levels of mRNA of the ASCT2Gene in Cell Lines, Tumor Mass of Xenografts, and Normal Tissues byReal-Time PCR Method (Quantitative PCR Method, or Q-PCR Method)

To 10 μL of each cDNA prepared in (3) (corresponding to 2 ng of polyA(+) RNA), a forward primer (Fw#1) for detecting a cDNA of the ASCT2gene comprising the nucleotide sequence represented by SEQ ID NO:3 whichwas designed from SEQ ID NO:1, and a reverse primer (Rv#1) for detectinga cDNA of the ASCT2 gene comprising the nucleotide sequence representedby SEQ ID NO:4 (all manufactured by Proligo) were added to give a finalconcentration of 300 nmol/L for each of them. Furthermore, to theresulting solution, 10×R-PCR buffer (Mg²⁺-free, manufactured by TakaraBio, 2 μL), 250 mmol/L Mg²⁺ solution (0.2 μL), dNTPs (10 mmol/L, 0.6μL), Ex Taq R-PCR (manufactured by Takara Bio, 0.2 μL) and SYBR Green I(manufactured by BMA; original solution product was diluted 2,500-fold,1 μL) were added. DEPC water was added thereto to give a total volume of20 μL.

PCR was carried out under the following reaction conditions: initiallyactivation of Taq DNA polymerase and denaturation of a template DNA at94° C. for 5 minutes, and then 45 cycles each consisting of threeprocesses, denaturation at 94° C. for 30 seconds, annealing at 65° C.for 30 seconds, and DNA elongation at 72° C. for 30 seconds. Thefluorescence intensity generated by SYBR Green I intercalated to theamplified product was measured by PRISM 7700 (manufactured by AppliedBiosystems) and data were analyzed according to the software, SequenceDetector ver. 1.7a, attached to the instrument PRISM 7700. Whether thesignal obtained by the above real-time PCR was the desired amplifiedfragment was judged by the size of the major amplified fragment obtainedby subjecting the reaction solution after completion of the reaction toagarose gel electrophoresis. The above real-time PCR was carried outusing a 96-well PCR plate. Besides the above cDNA-containing reactionsolution, a negative control (sterile water) and a sample for thepreparation of a calibration curve (1×10 to 1×10⁶ copies/well) preparedusing a plasmid HCHON2001712 (Homo sapiens cDNA FLJ43232 fis, The DNAData Bank of Japan (DDBJ) Accession No. AK125222) encoding a partialfragment of ASCT2 purified by a Qiagen Plasmid Midi Kit (manufactured byQiagen) were arranged as a sample in each well of the PCR plate and PCRwas then carried out.

FIGS. 1(1) to (4) show the alignment of a full-length sequence ofNM_(—)005628 (SEQ ID NO:1, GenBank Accession No. NM_(—)005628) which isa standard sequence of ASCT2 and the alignment of a full-length sequenceof plasmid HCHON2001712 which is used as a template for the preparationof a calibration curve. There are different sites between full-lengthsequences of NM_(—)005628 and HCHON2001712. Therefore, both of theforward primer (Fw#1) for detecting a cDNA comprising the nucleotidesequence represented by SEQ ID NO:3 and the reverse primer (Rv#1) fordetecting a cDNA comprising the nucleotide sequence represented by SEQID NO:4 which were used for the measurement of an ASCT2 expression levelwere designed using the region in which sequences of NM_(—)005628 andHCHON2001712 were completely identical therebetween.

Results of the expression level of mRNA of the ASCT2 gene in each of thethus obtained cell lines, xenograft tumor mass and normal tissues wereshown in FIGS. 2(1) to (2). The mRNA expression level was shown as arelative ratio in terms of numbers of expressed ASCT2 molecules per 2 ngof poly A(+) RNA and the ASCT2 gene-expression level in the airwayshowing the highest expression in normal tissues was defined as 1. Ascompared with a normal airway, enhanced expression levels wererecognized, that is, 10-fold or higher enhanced expression for bloodcancer-derived cell lines KG-1 (AML-derived cell line), HSB-2(ALL-derived cell line), K-562 (CML-derived cell line), and KMS-11 andARH-77 (both are MM-derived cell lines), 5-fold or higher enhancedexpression for Jurkat (ALL-derived cell line), IM-9, RPMI 8226 (both areMM-derived cell lines), HS-Sultan (BL-derived cell line), and ML-1(HL-derived cell line), and 2-fold or higher enhanced expression forTHP-1 (AML-derived cell line), CCRF-CEM (ALL-derived cell line), KU812(CML-derived cell line), Raji (BL-derived cell line), U-937 (HS-derivedcell line), gastric cancer tissues, and esophageal cancer tissues.

Example 2 Construction of CHO Cell Line Introducing Human ASCT2-myc/HisGene

In accordance with the following procedure, a plasmid pCR4-SLC 1A5-myc/His was obtained comprising the nucleotide sequence representedby SEQ ID NO:5 and the amino acid sequence represented by SEQ ID NO:6.Using this plasmid, a CHO cell line into which the human ASCT2-myc/Hisgene was introduced was obtained.

To the respective primers (10 pmol/L, each 1 μL) comprising each of thenucleotide sequences of SEQ ID NOs:7 to 10, 10×Ex Taq buffer (10 μL),dNTPs (2 mmol/L, 10 μL), and Ex Taq polymerase (1 μL, all manufacturedby Takara Bio) were added, and sterile water was further added theretoto give a total volume of 100 μL. In a manner similar to the proceduredescribed in Example 1(3), PCR was carried out under the followingreaction conditions: reaction at 96° C. for 2 minutes, and then 35cycles each consisting of three processes, reaction at 96° C. for 1minute, reaction at 60° C. for 1 minute, and reaction at 72° C. for 1minute. As a result, the nucleotide sequence (hereinafter, referred toas “N-SLC1A5”) was synthesized corresponding to the nucleotide sequenceat positions 1 to 370 when the nucleotide at a translation initiationpoint of the human ASCT2 gene was defined as position 1. The reactionproduct was separated by agarose gel electrophoresis. The resultingabout 0.4-kb amplified fragment was extracted using a QIAquick GelExtraction Kit (manufactured by Qiagen), and then cloned into a pCR4TOPOvector using a TOPO TA cloning kit (manufactured by Invitrogen)(hereinafter, the resulting plasmid is referred to as “pCR4-N-SLC1A5”).

Next, to 10 ng of the plasmid HCHON2001712 (DDBJ Accession No. AK125222)comprising the human ASCT2 gene as a template, the respective primers(10 μmol/L, each 1 μL) comprising the nucleotide sequences representedby SEQ ID NOs:11 and 12, 10×Ex Taq buffer (10 μL), dNTPs (2 mmol/L, 10μL), and Ex Taq polymerase (1 all manufactured by Takara Bio) wereadded, and sterile water was further added thereto to give a totalvolume of 100 μL. Under the same reaction conditions as described above,PCR was carried out to synthesize a nucleotide sequence (hereinafter,referred to as “C-SLC1A5-myc/His”) encoding a fusion protein having anaddition of myc/His to the C-terminal of a nucletotide sequencecorresponding to the nucleotide sequence of the human ASCT2 gene atpositions 365 to 1623. The reaction product was separated by agarose gelelectrophoresis. The resulting about 1.2-kb amplified fragment wasextracted using a QIAquick Gel Extraction Kit (manufactured by Qiagen).To the resulting extraction fragment as a template, the primers (10μmol/L, each 1 μL) comprising the nucleotide sequences represented bySEQ ID NOs: 11 and 13, 10×Ex Taq buffer (10 μL), dNTPs (2 mmol/L, 10μL), and Ex Taq polymerase (1 μL, all manufactured by Takara Bio) wereadded, and sterile water was further added thereto to give a totalvolume of 100 μL. Under the same reaction conditions as described above,PCR was carried out to add NotI and SpeI restriction enzyme sites to theC-terminal of C-SLC1A5-myc/His. The resulting reaction product wasseparated by agarose gel electrophoresis. The resulting about 1.2-kbamplified fragment was extracted using a QIAquick Gel Extraction Kit(manufactured by Qiagen), and then cloned into a pCR4TOPO vector using aTOPO TA cloning kit (manufactured by Invitrogen) (hereinafter, theresulting plasmid is referred to as “pCR4-C-SLC1A5-myc/His”). Theresulting gene sequence exhibited no amino acid variation even thoughthere was a substitution of T at position 1455 with C when a nucleotideat which a translation initiation point of the sequence represented bySEQ ID NO:1 was defined as position 1.

The resulting pCR4-N-SLC1A5 was digested with BssHII (manufactured byTakara Bio) and SpeI (manufactured by Takara Bio) and separated byagarose gel electrophoresis. The resulting about 4.4-kb gene fragmentwas extracted using a QIAquick Gel Extraction Kit (manufactured byQiagen). Similarly, the resulting pCR4-C-SLC1A5-myc/His was digestedwith BssHII (manufactured by Takara Bio) and SpeI (manufactured byTakara Bio), and an about 1.4-kb gene fragment was extracted. Eachextraction fragments were ligated using a Ligation high (manufactured byToyobo), and then Escherichia coli DH5α (manufactured by Toyobo) wastransformed with the resulting vector, in accordance with the method ofCohen et al [Proc, Natl, Acad-Sci. USA. 69 2110 (1972)]. A plasmid wasextracted from the resulting transformant using an automated plasmidisolation system PI-50 (manufactured by Kurabo), and a plasmidpCR4-SLC1A5 myc/His comprising the nucleotide sequence represented bySEQ ID NO:5 and the amino acid sequence represented by SEQ ID NO:6 wasobtained.

The resulting pCR4-SLC1A5 myc/His was digested with EcoRI (manufacturedby Takara Bio) and KpnI (manufactured by Takara Bio), and in a mannersimilar to the above, a gene fragment was extracted to obtain a fragmentcomprising a nucleotide sequence (hereinafter, referred to as“SLC1A5-myc/His”) encoding a fusion protein in which myc/His was addedto the C-terminal of the human ASCT2 gene.

The resulting fragment containing SLC 1 A5-myc/His was ligated into apKANTEX93 vector (WO97/10354) which had been previously digested withEcoRI (manufactured by Takara Bio) and KpnI (manufactured by TakaraBio). In a manner similar to the above, Escherichia coli DH5α(manufactured by Toyobo) was transformed with the ligation product.After obtaining a transformant, an expression plasmidpKANTEX-SLC1A5-myc/His was obtained using a plasmid isolation kit(manufactured by Qiagen).

In accordance with electroporation [Cytotechnology, 3, 133 (1990)],introduction of pKANTEX-SLC1A5-myc/His into a CHO/DG44 cell [SomaticCell and Molecular Genetics. 12, 555 (1986)] was carried out in thefollowing manner. The cells used herein were those subcultured in amedium where 1×HT supplement (manufactured by Invitrogen) was added toIMDM (manufactured by Invitrogen) containing 10% FBS (manufactured byLife Technologies) and gentamicin (50 μg/mL, manufactured by NacalaiTesque) (hereinafter, referred to as “A3 medium”). CHO/DG44 cells weresuspended in buffer containing potassium chloride (137 nmol/L), sodiumchloride (2.7 nmol/L), disodium hydrogen phosphate (8.1 mmol/L), sodiumdihydrogen phosphate (1.5 nmol/L) and magnesium chloride (4 mmol/L)(hereinafter, referred to as “K-PBS”) to give a cell density of 8×10⁶cells/mL, and the resulting cell suspension (200 μL, 1.6×10⁶ cells interms of cell count) was mixed with an expression plasmidpKANTEX-SLC1A5-myc/His (10 μg). The mixture was transferred into acuvette (interelectrode distance: 2 mm), and gene introduction wascarried out using a GenePulser II (manufactured by Bio-Rad) at a pulsevoltage of 0.35 kV and an electric capacity of 250 μF. The cuvette wasallowed to stand on ice, and a cell suspension in the cuvette wassuspended in a cell culture vessel containing an A3 medium and culturedin a 5% CO₂ incubator at 37° C. Four days after the culturing, theculture medium was exchanged with an A3 medium containing G418(manufactured by Nacalai Tesque; 0.5 mg/mL), followed by cell culture.With medium exchange and subculturing during the cell culture, atransformed cell resistant to G418 was obtained about two weeks afterthe gene introduction.

The resulting G418-resistant transformed cells were diluted to give acell density of 5 cells/10 mL in an A3 medium containing 0.5 mg/L ofG418 (manufactured by Nacalai Tesque), and 100 μL/well of the dilutedcell suspension was dispensed into a 96-well plate, followed by growingin stepwise increasing concentrations of methotrexate. In this manner, aclone showing a high expression level of ASCT2-myc/His was selected,from which a CHO cell line with the introduction of human ASCT2-myc/Hisgene was then obtained.

The resulting human ASCT2-myc/His gene-introduced CHO cells (1×10⁵ to5×10⁵ cells) were suspended in 70% ethanol-PBS (1 mL) and fixed at icetemperature for 30 minutes. After dispensing at 1×10⁶ to 5×10⁶cells/well into a 96-well U-bottom plate and subsequent centrifugationat 1,500 rpm for 5 minutes, the supernatant was discarded, followed byblocking with 1% BSA-PBS at ice temperature for 30 minutes. Afterremoving the supernatant by centrifugation, an anti-myc antibody PL14(manufactured by Medical & Biological Laboratories), an anti-Hisantibody (manufactured by Qiagen) and a mouse IgG1 isotype control(manufactured by Dako), as the primary antibodies, were diluted with 1%BSA-PBS to give final concentrations of 1.0, 0.1 and 0.1 μg/mL,respectively, and dispensed at 100 μL/well to react at ice temperaturefor 60 minutes. After the plate was washed once with 1% BSA-PBS, 100μL/well of an FITC-labeled anti-mouse immunoglobulin G (H+L)(manufactured by DAKO) diluted 50-fold with 1% BSA-PBS, as the secondaryantibody, was added and react at ice temperature for 30 minutes undershading. After washing once again with 1% BSA-PBS, the cells weresuspended in PBS and the fluorescence intensity was measured by a flowcytometer (Cytomics FC500MPL, manufactured by Beckman Coulter). Theresults were shown in FIG. 3. From the fact that a high reactivity wasdetected in the anti-myc antibody and the anti-His antibody, it wasconfirmed that a desired CHO cell line with the introduction of thehuman ASCT2-myc/His gene was constructed.

Example 3 Construction of Monoclonal Antibody Against N-Terminal PartialPeptide of ASCT2

(1) Preparation of Immunogen

For binding to a carrier protein, an N-terminal partial peptide of thehuman ASCT2 gene represented by SEQ ID NO:14 (amino acid residues atpositions 2 to 16 from the N-terminal) in which Cys was added to theC-terminal was synthesized using an automated synthesizer (PSSM-8,manufactured by Shimadzu).

In order to enhance the immunogenity, a conjugate with KLH (manufacturedby Wako Pure Chemical) was prepared as follows and used as an immunogen.That is, KLH was dissolved in PBS to give a concentration of 10 mg/mLand a 1/10 volume of N-(m-maleimide benzoyloxy)succinimide (MBS,manufactured by Nacalai Tesque, 25 mg/mL) was then added theretodropwise, followed by reaction under stirring for 30 minutes. Thereaction solution was passed through a gel filtration column (SephadexG-25 column, manufactured by GE Healthcare) which had been previouslyequilibrated with PBS, and non-reacted MBS was removed to obtainKLH-MBS. The N-terminal partial peptide of human ASCT2 to which Cys wasadded (1 mg) was dissolved in sodium phosphate buffer (0.1 mol/L, pH7.0) and 2.5 mg of KLH-MBS was added thereto, followed by reaction understirring at room temperature for 3 hours. After the reaction wascomplete, the reaction solution was dialyzed against PBS, therebyobtaining a human ASCT2 N-terminal peptide-KLH conjugate as animmunogen.

(2) Immunization of Animals and Preparation of Antibody-Producing Cell

The human ASCT2 N-terminal peptide-KLH conjugate (100 μg) obtained inthe above (1) together with 2 mg of aluminum gel and 1×10⁹ cells of apertussis vaccine (manufactured by Chiba Serum Institute) wasadministered to 4-week old female SD rats (manufactured by Japan SLC).Two weeks after the first administration, the conjugate (100 μg) wasadditionally administered to the rats once a week, four times in total.Blood was collected from caudal veins of the rats, and the reactivitythereof with the human ASCT2 partial peptide was investigated by thefollowing enzyme immunoassay. Three days after the final immunization,the spleen was excised from the rat which showed sufficient antibodytiter.

The spleen was minced into small pieces in MEM (manufactured by NissuiPharmaceutical), loosened with forceps, and centrifuged at 1,200 rpm for5 minutes (CR5B, manufactured by Hitachi). To the obtained precipitatedfraction, Tris-ammonium chloride buffer (pH 7.65) was added and reactedfor 1 to 2 minutes, whereby red blood cells were removed. The cellfraction obtained as a precipitation fraction was washed three timeswith MEM, thereby preparing antibody-producing cells.

(3) Enzyme Immunoassay

As an assay antigen, the Cys-added N-terminal partial peptide of humanASCT2 presented by SEQ ID NO:14 was prepared in the form of a conjugatewith thyroglobulin (hereinafter, referred to as “THY”), according to thefollowing procedure. The construction method of the conjugate was thesame as in (1), but succinimidyl 4-[N-maleimidemethyl]-cyclohexane-1-carboxylate (SMCC, manufactured by Sigma) was usedinstead of MBS.

The resulting human ASCT2 N-terminal peptide-THY conjugate (10 μg/mL, at50 μL/well) was dispensed into a 96-well EIA plate (manufactured byGreiner), and allowed to stand at 4° C. overnight for adsorption. Afterthe non-adsorbed conjugate was washed, 1% BSA-PBS (100 μL/well) wasadded to the plate, followed by reaction at room temperature for 1 hour,and the remaining active groups were blocked. After the non-reactedBSA-PBS was washed, 50 μL/well of the test material such as antiserum orculture supernatant, as the primary antibody, was dispensed to theplate, followed by reaction for 2 hours. The plate was washed with 0.05%Tween-PBS and 50 μL/well of a diluted peroxidase-labeled anti-ratimmunoglobulin (manufactured by Dako) as the secondary antibody wasadded to the plate, followed by reaction at room temperature for 1 hour.After the plate was washed with 0.05% Tween-PBS, a 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)ammonium (ABTS) substratesolution [ABTS (manufactured by Wako Pure Chemical, 1 mmol/L), citratebuffer (0.1 mol/L, pH 4.2), and H₂O₂ (0.1%)] was added to the well andthen color-developed. An absorbance (OD415-OD490) at a sample wavelengthof 415 nm and then a reference wavelength of 490 nm was measured using aplate reader (Emax microplate reader, Molecular Devices).

(4) Preparation of Mouse Myeloma Cell

The 8-azaguanine-resistant mouse myeloma cell line P3-U1 [P3X63Ag8U.1,ATCC Accession No. CRL-1597, European Journal of Immunology, 6, 511(1976)] was cultured in a medium (hereinafter referred to as “normalmedium”) where glutamine (1.5 mmol/L), 2-mercaptoethanol (5×10⁻⁵ mol/L),gentamicin (10 μg/mL) and FBS (10%) were added to an RPMI 1640 medium(manufactured by Invitrogen) to ensure the cell count of 2×10⁷ or morenecessary for cell fusion, and was provided as parent cells in the cellfusion.

(5) Preparation of Hybridoma

The antibody-producing cells obtained in (2) and the myeloma cellsobtained in (4) were mixed at a ratio of 10:1 and the obtained cellswere centrifuged at 1,200 rpm for 5 minutes (CR5B, manufactured byHitachi). The supernatant was discarded and the precipitated cells werewell loosened. A mixture of PEG 1000 (1 g), MEM (manufactured byInvitrogen, 1 mL) and dimethyl sulfoxide (0.35 mL) was added thereto at0.5 mL/1×10⁸ antibody-producing cells and at 37° C. under stirring, andMEM (1 mL) was added several times to the suspension every 1 to 2minutes and then MEM was added to give a total volume of 50 mL. Aftercentrifugation at 900 rpm for 5 minutes (CR5B, manufactured by Hitachi),the supernatant was discarded and the precipitated cells were slowlyloosened. Then, the cells were suspended in 100 mL of a normal medium towhich HAT Media Supplement (manufactured by Invitrogen) was added(hereinafter, referred to as “HAT medium”) by gently pipetting up anddown.

Into a 96-well culture plate, 200 μL/well of the obtained suspension wasdispensed and cultured in a 5% CO₂ incubator for 10 to 14 days at 37° C.

After culturing, the culture supernatant was examined by the enzymeimmunoassay described in (3), the wells which specifically reacted tothe N-terminal partial peptide of human ASCT2 were selected. The cellscontained in the selected wells were subjected to cloning by a limitingdilution method twice to give a hybridoma KM3842 which produces amonoclonal antibody against the N-terminal partial peptide of ASCT2. Theantibody subclass of KM3842 was determined to be rat IgG2a by using asubclass typing kit (manufactured by ABT Sarotech) (FIG. 4).

(6) Obtaining of Purified Monoclonal Antibody

The hybridoma (5×10⁶ to 20×10⁶ cells/animal) obtained in (5) wasintraperitoneally injected into pristane-treated 8-week-old female nudemice (Balb/c, manufactured by Japan SLC). After 10 to 21 days, thehybridoma became to ascites carcinoma. The ascitic fluid (1 to 8mL/animal) was collected from the mice which had produced ascites. Then,the ascites was centrifuged at 3,000 rpm for 5 minutes (CR5B,manufactured by Hitachi) to remove solids. The resulting solution waspurified by the caprylic acid precipitation method [Antibodies-ALaboratory Manual, Cold Spring Harbor Laboratory (1988)] to obtain apurified KM3842 antibody.

Example 4 Investigation of Reactivity of N-Terminal Partial Peptide ofASCT2 with Purified Monoclonal Antibody

The reactivity of a monoclonal antibody KM3842 with the N-terminalpartial peptide of ASCT2 was examined in accordance with the enzymeimmunoassay described in Example 3(3). The purified antibody KM3842obtained in Example 3(6) was diluted with 1% BSA-PBS to giveconcentrations of 10, 1, 0.1, 0.01, 0.001, and 0.0001 μg/mL,respectively, and used as a primary antibody. As a result of themeasurement, as shown in FIG. 5, the antibody KM3842 exhibited aspecific reactivity with the N-terminal partial peptide of ASCT2.

Example 5 Construction of Monoclonal Antibody Against ExtracellularRegion of ASCT2

(1) Preparation of immunogen

The human ASCT2-myc/His gene-introduced CHO cell line obtained inExample 2 was cultured in IMDM (manufactured by Invitrogen) containing10% FBS for 2 to 3 days and was peeled off using a 0.02%ethylenediaminetetraacetic acid (EDTA) solution (manufactured by NacalaiTesque). The cells were suspended in PBS to give a cell count of 6×10⁶to 1×10⁷ cells per one immunized animal.

(2) Immunization of Animal and Preparation of Antibody-Producing Cell

The cell suspension obtained in (1) was administered to 6-week old maleBXSB mice (n=3/group, manufactured by Japan SLC) or 4-week old female SDrats (n=3/group, manufactured by Japan SLC) together with 1×10⁹ cells ofa pertussis vaccine (manufactured by Chiba Serum Institute). One weekafter the administration, the cell suspension was administered to theanimals once a week, four times in total. Thereafter, blood wascollected from the fundus oculi of mice or caudal veins of rats. Theantibody titer thereof in the blood was measured by a fluorescent cellstaining method using the following cell-based assay system (ABI 8200Cellular Detection System, manufactured by Applied Biosystems) or flowcytometer (Cytomics FC500 MPL, manufactured by Beckman Coulter). Threedays after the final immunization, the spleen was excised from a mouseor rat which showed sufficient antibody titer.

In the same manner as Example 3(2), the antibody-producing cells wereprepared from the obtained spleen.

(3) Fluorescent Cell Staining Method—1 (Cell-Based Assay System)

The ASCT2-myc/His gene-introduced CHO cell line obtained in Example 2and the vector-introduced CHO cells were used as assay cells. Each cellwere cultured in IMDM (manufactured by Invitrogen) containing 10% FBSfor 2 to 3 days and peeled off with a trypsin-EDTA solution(manufactured by Invitrogen) was suspended in the same medium, seededinto an ABI8200 black 96-well plate at a cell density of 1×10⁴ cells/100μL medium/well, and cultured overnight. The test substance such asantiserum or culture supernatant (10 μL/well) was dispensed to the plateas the primary antibody, 100 μL/well of ALEXA647-labeled anti-mouseimmunoglobulin G (H+L) or ALEXA647-labeled anti-rat immunoglobulin G(H+L) (all manufactured by Invitrogen) was then added as the secondaryantibody, followed by allowing to stand for 4 hours under shading.Fluorescence of 650 to 685 nm excited with a laser (633 nm He/Ne) wasmeasured by the ABI 8200 Cellular Detection System (manufactured byApplied Biosystems).

(4) Fluorescent Cell Staining Method—2 (Flow Cytometer)

The human ASCT2-myc/His gene-introduced CHO cell line obtained inExample 2 and vector-introduced CHO cells were used as assay cells. Eachcells which were cultured in IMDM (manufactured by Invitrogen)containing 10% FBS for 2 to 3 days and peeled off with a 0.02% EDTAsolution (manufactured by Nacalai Tesque) were washed with PBS and wereblocked for 20 minutes at ice temperature using 1% BSA-PBS in order toavoid the non-specific adsorption of antibodies. The resulting cellswere seeded into a 96-well U-bottom plate at cell density of 5×10⁵cells/50 μL/well, followed by centrifugation at 1,800 rpm for 2 minutes(05PR-22, manufactured by Hitachi Koki), and then the supernatant wasremoved. The test substance (50 μL/well) such as antiserum or culturesupernatant was dispensed as the primary antibody, followed by reactionat ice temperature for 30 minutes. Washing was carried out 3 times by acentrifugation method using PBS and 50 μL/well of ALEXA 488-labeledanti-mouse immunoglobulin G (G+L) or ALEXA488-labeled anti-ratimmunoglobulin G (G+L) (all manufactured by Invitrogen) was added as thesecondary antibody, followed by reaction at ice temperature for 30minutes under shading. After the cells were washed again using PBS threetimes by centrifugation, the cells were suspended in PBS, and thefluorescence of 510 to 530 nm excited with a 488 nm Ar laser wasmeasured by a flow cytometer (Cytomics FC500MPL, manufactured by BeckmanCoulter).

(5) Construction of hybridoma

In the Same Manner as Example 3(5), the Cell Fusion was Carried Outbetween the antibody-producing cell obtained in (2) and the myeloma cellobtained in Example 3(4).

Next, the cells obtained from the cell fusion were suspended in an HATmedium. Into a 96-well culture plate, 200 μL/well of the suspension ofthe resulting cell was dispensed and the cells were cultured in a 5% CO₂incubator for 8 to 10 days at 37° C.

The reactivity of the post-culture supernatant was confirmed by thefluorescent cell staining method described in the above (3) and (4) andthe well which reacts with the human ASCT2-myc/His gene-introduced CHOcell line and does not react with the vector-introduced CHO cells wasselected. Then, the cells contained in the selected wells were subjectedto cloning by a limiting dilution method twice to give hybridomasKM3998, KM4000, KM4001, KM4008, KM4012, and KM4018 which producemonoclonal antibodies against the extracellular region of ASCT2.

The determination of the subclass of a mouse monoclonal antibody amongthe obtained hybridomas was carried out in accordance with the followingprocedure.

Anti-mouse immunoglobulin rabbit polyclonal antibodies (manufactured byDako, 10 μg/mL, 50 μL/well) was dispensed into a 96-well EIA plate(manufactured by Greiner) and allowed to stand at 4° C. overnight foradsorption. After the non-absorbed conjugate was washed, 100 μL/well of1% BSA-PBS was added to the plate, followed by reaction at roomtemperature for 1 hour in order to block the remaining active groups.After the non-reacted BSA-PBS was washed, 50 pt/well of the testmaterial was dispensed to the plate, followed by reaction for 2 hours.The plate was washed with 0.05% Tween-PBS and a dilutedsubclass-specific peroxidase-labeled anti-mouse immunoglobulin(manufactured by Invitrogen; 50 μL/well) was added to the plate as thesecondary antibody, followed by reaction at room temperature for 1 hour.The plate was washed with 0.05% Tween-PBS and color-developed by addingthe ABTS substrate solution used in Example 3(3). Then, an absorbance(OD415-OD490) at a sample wavelength of 415 nm and a referencewavelength of 490 nm was measured using a plate reader (Emax microplatereader, Molecular Devices). The subclass of the mouse monoclonalantibody whose subclass could not be determined by the above-mentionedmethod was determined using a mouse rat monoclonal isotyping kit(manufactured by Dainippon Sumitomo Pharma). Further, the subclass ofthe rat monoclonal antibody was determined using a rat monoclonalisotyping kit (manufactured by Dainippon Sumitomo Pharma).

FIG. 4 shows the list of the subclass of the antibody derived fromindividual hybridomas.

(6) Obtaining of Purified Monoclonal Antibodies—1

Purified antibodies of KM3998 were obtained in accordance with thefollowing procedure.

The hybridoma (5×10⁶ to 20×10⁶ cells/animal) obtained in (5) wasintraperitoneally injected into pristane-treated 8-week-old female nudemice (Balb/c, manufactured by Japan SLC). The ascitic fluid (1 to 8mL/animal) was collected from the mice when the hybridoma developedascites tumor in 10 to 21 days. Then, the ascites was subjected tofiltration [5 μm, polyethersulfone (PES) membrane, manufactured by Pall]to remove solids, followed by purification with the caprylic acidprecipitation method [Antibodies-A Laboratory Manual, Cold Spring HarborLaboratory (1988)].

(7) Obtaining of Purified Monoclonal Antibodies—2

Each of the purified antibodies of KM4000, KM4001, KM4008, KM4012 andKM4018 were obtained in accordance with the following procedure.

The individual hybridomas obtained in the above (5) were cultured inRPMI 1640 containing 10% FBS in a 5% CO₂ incubator at 37° C. At thepoint of the cell count of 5×10⁷ cells, the supernatant was discarded bycentrifugation at 1,200 rpm for 5 minutes (CR5B, manufactured byHitachi). The resulting cells were suspended at a cell density of 1×10⁵cells/mL in a Hybridoma-SFM (manufactured by Gibco) comprising 5%Daigo's GF21 (manufactured by Wako Pure Chemical) to give a volume of500 mL and then cultured in a 5% CO₂ incubator for three days at 37° C.The resulting cell suspension was centrifuged at 1,500 rpm for 5 minutesand the resulting supernatant was filtered through a bottle top filter(0.2 μm, PES membrane, manufactured by Corning).

Purification was carried out using a Protein A-conjugated resin(manufactured by Millipore) for KM4000, KM4001, KM4008 and KM4012 andusing a Protein G-conjugated resin (manufactured by Millipore) forKM4018. Each resin was packed into a 1 mL mini-column and 10 mL ofequilibration buffer was passed through it to achieve equilibration. Theequilibration buffer to be used was glycine (1 mol/L)-sodium chloride(0.15 mol/L) (pH 8.6) for the Protein A-conjugated resin and glycine(0.5 mol/L)-PBS (pH 7.4) for the Protein G-conjugated resin. The culturesupernatant which was obtained by the filtration treatment was passedthrough the column at a flow rate of about 100 mL/h and then the columnwas washed with 10 mL of the equilibration buffer. Thereafter, theantibodies adsorbed to the column were eluted with citrate buffer (0.1mol/L, pH 3.0). Into tubes in which 80 μL of Tris (2 mol/L, pH 8.0) hadbeen previously filled, and then 500 μL/tube of the eluted antibodieswere aliquoted. Then, an absorbance (OD280 nm) of each tube was measuredusing a UV-V spectrophotometer (UV-1600, manufactured by Shimadzu) andthe fraction in which the protein was detected was recovered. Afterovernight dialysis in PBS at 4° C., the fraction was filtrated (0.2 μm,PES membrane, manufactured by Pall) and the antibody concentration wascalculated from the absorbance at 280 nm (OD280 nm) [OD280 nmmeasurement value (A)/1.4=protein concentration (mg/mL)].

Example 6 Evaluation of Reactivity of Monoclonal Purified Antibodieswith Extracellular Region of ASCT2

(1) Fluorescent Cell Staining Method (Flow Cytometer)

The human ASCT2-myc/His gene-introduced CHO cell obtained in Example 2,the vector-introduced CHO cell and the multiple myeloma cell line KMS-11(HSRRB No. JCRB1179) were respectively cultured in a 5% CO₂ incubatorfor 3 to 4 days at 37° C. The human ASCT2-myc/His gene-introduced CHOcells and the vector-introduced CHO cells were peeled off with a 0.02%EDTA solution (manufactured by Nacalai Tesque) and washed with a mixedsolution of PBS, 0.02% EDTA and 0.05% sodium azide. Additionally, inorder to avoid the non-specific adsorption of antibodies, the cells wereblocked for 30 minutes at ice temperature using 1% BSA-PBS for the humanASCT2-myc/His gene-introduced CHO cells and the vector-introduced CHOcells, and using 100 μg/mL of human IgG (manufactured by Sigma) forKMS-11. The cells were seeded into a 96-well U-bottom plate so as togive a density of 1×10⁵ to 5×10⁵ cells/100 μL/well and centrifuged at1,500 rpm for 5 minutes (05PR-22, manufactured by Hitachi Koki) and thenthe supernatant was removed. The test substances as the primaryantibody, i.e., the purified antibodies and rat IgG2a-UNLB (negativecontrol, manufactured by Beckman Coulter) or KM511 {negative control,anti-G-CSF derivative antibody, [Agric. Biol. Chem., 53, 1095 (1989)],mouse IgG1} were diluted to give a final concentration of 10 μg/mL in amixed solution (hereinafter, referred to as “Dilution Solution A”) of 1%BSA-PBS, 0.02% EDTA and 0.05% sodium azide and the resulting solutionwas added at the volume of 100 μL/well, followed by reaction for 60minutes at ice temperature. After washing twice with the DilutionSolution A, 100 μL/well of ALEXA488-labeled anti-mouse immunoglobulin G(G+L) (manufactured by Invitrogen) or 100 μL/well of ALEXA488-labeledanti-rat immunoglobulin G (G+L) (manufactured by Invitrogen) or 100μL/well of an FITC-labeled anti-mouse kappa-chain antibody (manufacturedby Southern Biotech) or an FITC-labeled anti-rat kappa-chain antibody(manufactured by Southern Biotech), diluted with Dilution Solution A,was added as the secondary antibody, followed by reaction for 30 minutesat ice temperature under shading. Washing with Dilution Solution A wascarried out three times again, the cells were suspended in PBS and thefluorescence intensity was measured by a flow cytometer (CytomicsFC500MPL, manufactured by Beckman Coulter).

The measurement results are shown in FIGS. 6 and 7.

As shown in FIG. 6, KM13998 exhibited a high binding activity to thehuman ASCT2-myc/His gene-introduced CHO cells and KMS-11 expressingASCT2mRNA, but no binding activity to the vector-introduced CHO cells.The negative control antibody rat IgG2a-UNLB exhibited no bindingactivity for any type of cell. From these results, it was demonstratedthat KM3998 has a specific binding activity for ASCT2-expressing cells.

Further, as shown in FIG. 7, KM4000, KM4001, KM4008, KM4012 and KM4018exhibited a potent binding activity (mean fluorescence intensity) to thehuman ASCT2-myc/His gene-introduced CHO cells and theASCT2-mRNA-expressing KMS-11, but exhibited no binding activity to thevector-introduced CHO cells. In addition, the negative controlantibodies, i.e., rat IgG2a-UNLB and KM511 exhibited no binding activityfor any type of cells. From these results, it was demonstrated thatKM4000, KM4001, KM4008, KM4012, and KM4018 has a specific bindingactivity to ASCT2-expressing cells.

(2) Western Blotting

To each of the human ASCT2-myc/His gene-introduced CHO cell obtained inExample 2, the vector-introduced CHO cell, the multiple myeloma cellline KMS-11 (HSRRB No. JCRB1179) and the colorectal cancer cell lineWiDr (ATCC Accession No. CCL-218), a mixed solution (hereinafter,referred to as “cell lysis buffer A”) of Tris-HCl (50 mmol/L, pH 7.2),1% Triton X-100, sodium chloride (150 mmol/L), magnesium chloride (2mmol/L), calcium chloride (2 mmol/L), 0.1% sodium azide,phenylmethanesulfonyl fluoride (PMSF, 5 μmol/L), N-ethyl maleimide (50mmol/L), leupeptin (1 mg/mL) and dithiothreitol (0.1 mmol/L) was addedat a volume of 1 mL per 5×10⁷ cells, and the resulting solution wasallowed to stand at 4° C. for 2 hours. The solution was centrifuged toobtain the supernatant which was used as a cell lysate. Each of celllysates of 5×10⁴ cells/lane was fractionated using SDS-polyacrylamideelectrophoresis (PAGEL, manufactured by Atto) and transferred to a PVDFmembrane (manufactured by Millipore). After the transferred PVDFmembrane was blocked with 10% BSA-PBS, each of the test substances asthe primary antibody, i.e., the purified antibodies, KM3842 (positivecontrol) obtained in Example 3, rat IgG2a-UNLB (negative control,manufactured by Beckman Coulter) and KM511 (negative control) wasdiluted to give a concentration of 10 μg/mL in 1% BSA-PBS, followed byreaction at 4° C. overnight. The resulting PVDF membrane was thoroughlywashed with 0.1% Tween-PBS (hereinafter, referred to as “PBST”),followed by reaction with the secondary antibody, i.e.,peroxidase-labeled anti-mouse immunoglobulin G (H+L) (manufactured byZymed) or peroxidase-labeled anti-rat immunoglobulin G (H+L)(manufactured by Dako) for 1 hour at room temperature. The PVDF membranewas thoroughly washed again with PBST and the antibody-bound band wasdetected using ECL Western Blotting Detection Reagents (manufactured byAmersham Pharmacia).

KM3842 could detect a band around a molecular weight of 75 kDacorresponding to a molecular weight of ASCT2 in the human ASCT2-myc/Hisgene-introduced CHO cells, ASCT2 mRNA-expressing KMS-11 cells and WiDrcells, but KM3998, KM4000, KM4001, KM4008, KM4012 and KM4018 could notdetect ASCT2. Further, the reactivity of any antibody was not recognizedagainst the vector-introduced CHO cells.

From the results of the above flow cytometry and Western blotting, it isconsidered that the each of binding activity of KM3998, KM4000, KM4001,KM4008, KM4012 and KM4018 to ASCT2 was lost by SDS denaturation of ASCT2and it was demonstrated that KM3998, KM4000, KM4001, KM4008, KM4012 andKM4018 were the antibodies which recognize and bind to the nativethree-dimensional structure of ASCT2.

(3) Immunoprecipitation Method

To each of the human ASCT2-myc/His gene-introduced CHO cell obtained inExample 2 and the vector-introduced CHO cells, a mixed solution(hereinafter, referred to as “cell lysis buffer B”) of Tris-HCl (50mmol/L, pH 7.5), 1% Triton X-100, sodium chloride (150 mmol/L), EDTA (5mmol/L), 0.1% SDS, 0.5% sodium deoxycholate, Protease inhibitor cocktail(manufactured by Roche Diagnostics) and Phosphatase inhibitor cocktail(manufactured by Roche Diagnostics) was added at a volume of 1 mL per2×10⁷ cells and the resulting solution was stirred at 4° C. for 30minutes, followed by centrifugation (CF15D, manufactured by HitachiKoki). A protein concentration of the resulting supernatant was measuredusing a protein assay reagent (manufactured by Bio-Rad) and adjusted to5 mg/mL by cell lysis buffer B to be used as a cell lysate.

Then, 2 μg of each test substance, i.e., the purified antibody, KM3842(positive control) obtained in Example 3, rat IgG2a-UNLB (negativecontrol, manufactured by Beckman Coulter) and KM511 (negative control),was mixed with 1 mg of the resulting cell lysate at 4° C. for 1 hour.

With 0.1% BSA-PBST (300 μL), Protein G-Sepharose beads (manufactured byAmersham; 30 μL) or Protein A-Sepharose beads (manufactured by Amersham;30 μL) was pre-treated for 30 minutes or more, followed bycentrifugation to remove the supernatant and the beads were suspended ina mixed solution (hereinafter, referred to as “bead wash buffer”) (90μL) of Tris-HCl (50 mmol/L, pH 7.5), 1% Triton X-100, sodium chloride(150 mmol/L), EDTA (5 mmol/L) and Protease inhibitor cocktail(manufactured by Roche Diagnostics).

To the bead suspension, a mixed solution (100 μL) of antibodies and celllysate was added and mixed at 4° C. for 2 hours and then the beads wererecovered by centrifugation. The recovered beads were washed three tofive times with the bead wash buffer and dissolved in a mixed solution(hereinafter, referred to as “SDS-PAGE sample buffer”) of 2% SDS,Tris-HCl (62 mmol/L, pH 6.8) and 10% glycerol. The resulting solutionwas analyzed by the following immunoblotting.

The obtained solution was fractionated by SDS-polyacrylamideelectrophoresis and transfered to a PVDF membrane (manufactured byMillipore).

The resulting PVDF membrane was blocked with 5% skim milk-PBST andallowed to react with 3.5 μg/mL of KM3842 (positive control) obtained inExample 3 as a primary antibody for 2 hours at room temperature. Theresulting PVDF membrane was thoroughly washed with PBST and allowed toreact with peroxidase-labeled anti-rat immunoglobulin G (H+L)(manufactured by Dako) at room temperature for 1 hour. The PVDF membranewas thoroughly washed again with PBST and the antibody-bound band wasdetected using ECL Western Blotting Detection Reagents (manufactured byAmersham Pharmacia).

KM4000, KM4001, KM4008, KM4012, KM4018 and positive control antibodyKM3842 could detect a band around a molecular weight of 75 kDa. As aresult, it was demonstrated that KM4000, KM4001, KM4008, KM4012 andKM4018 are antibodies which can detect ASCT2 by the immunoprecipitationreaction and recognize the three-dimensional structure of ASCT2.

(4) Inhibitory activity on intracellular uptake of amino acids by ASCT2

The human colorectal cancer cell line WiDr (ATCC Accession No. CCL-218)was adjusted to give a cell density of 2×10⁴ cells/mL in a Dulbecco'smodified Eagle's medium (DMEM, manufactured by Invitrogen) containing10% inactivated dialyzed fetal bovine serum (hereinafter, referred to asdFBS, manufactured by Invitrogen) (hereinafter, referred to as“glutamine-free medium”) and 100 μL/well of the resulting supension wasseeded into a 96-well plate, followed by culturing in a 5% CO₂ incubatorat 37° C. for 24 hours.

Each test substance, i.e., purified antibody, rat IgG2a-UNLB (negativecontrol, manufactured by Beckman Coulter), KM511 (negative control) or aglutamine-competitive amino acid mixture [AST mixture, a mixture ofalanine, serine and threonine (all manufactured by Sigma), 3.3 mmol/Lfor each)], was diluted with PBS to give a final concentration of 31.6to 0.03 μg/mL. After, 20 μL/well of the resulting solution was added tothe well, 20 μL/well of a glutamine solution (manufactured byInvitrogen) which was prepared to have a final concentration of 0.2mmol/L in a glutamine-free medium was added. To the wells of the controland the wells of the blank plate, PBS (20 μL/well) and the aboveglutamine solution (20 μL/well) were added. After addition ofantibodies, the plates except for the blank plate were incubated in a 5%CO₂ incubator at 37° C. for 72 hours.

Furthermore, 20 μL/well of{4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate}diluted to 50% in a glutamine-free medium (hereinafter, referred to as“WST-1 reagent”, manufactured by Roche Diagnostics) was added thereto,followed by further incubation at 37° C. for 2 hours.

An absorbance at 450 nm (control wavelength 650 nm) was measured using amicroplate spectrophotometer (Emax microplate reader, manufactured byMolecular Devices). A relative proliferation rate (%) of theantibody-added well was calculated by regading an absorbance of thenon-antibody added control well as 100% and an absorbance of the blankplate well as 0%.

The measurement results were shown in FIGS. 8 and 9.

As shown in FIG. 8, KM3998 exhibited slight inhibition of cellproliferation at a high concentration, but its inhibitory effect waslower than that of the glutamine-competitive amino acid mixture.Therefore, it was found that a neutralizing activity of KM3998 on ASCT2was low.

Further, as shown in FIG. 9, all of KM4000, KM4001, KM4008, KM4012 andKM4018 strongly inhibited the cell proliferation depending on anantibody concentration. As a result, it was demonstrated that KM4000,KM4001, KM4008, KM4012 and KM4018 strongly neutralize the function ofASCT2 for intracellular uptake of glutamine and consequently exhibit asignificant inhibitory activity against the proliferation of cancercells.

Example 7 Isolation and Analysis of cDNA Encoding Variable Regions ofAnti-ASCT2 Monoclonal Antibody

(1) Preparation of mRNA from Anti-ASCT2 Monoclonal Antibody-ProducingHybridoma Cell

From 5×10⁷ cells of the respective hybridomas KW4008, KM4012 and KM4018obtained in Example 5(5), about 6 μg of mRNA was prepared using anRNeasy Maxi Kit (manufactured by Qiagen) and an Oligotex-dT30<Super>mRNAPurifiction Kit (manufactured by Takara Bio) in accordance with theinstructions attached thereto.

(2) Gene Cloning of H Chain and L Chain Variable Regions of Anti-ASCT2Monoclonal Antibody

Using a BD SMART RACE cDNA Amplification Kit (manufactured by BDBiosciences) in accordance with the instructions attached thereto, cDNAseach comprising the nucleotide sequence of BD SMART II A Oligonucleotideattached to the kit at the 5′-terminal were obtained from 0.6 μg of mRNAof KM4008, KM4012 and KM4018 obtained in (1).

The resulting cDNA was used as a template and PCR was carried out usingthe universal primer A mix attached to the kit and the mouseIg(γ)-specific primer (mG3a2 or mG2bal) represented by SEQ ID NOs:15 and16 or the rat Ig(γ)-specific primer (rG2a) represented by SEQ ID NO:43so that the cDNA fragment of VH was amplified. Another PCR was carriedout using the mouse Ig(κ)-specific primer (mKa1) represented by SEQ IDNO:17 or the rat Ig(κ)-specific primer (rKa2) represented by SEQ IDNO:44 in place of the Ig(γ)-specific primer to amplify the cDNA fragmentof VL.

PCR for the rat Ig(κ)-specific primer (rKa2) was carried out by heatingat 94° C. for 5 minutes; 40 cycles each consisting of reaction at 94° C.for 15 seconds, reaction at 68° C. for 30 seconds and reaction at 72° C.for 3 minutes; and then reaction at 72° C. for 10 minutes. Other PCR wascarried out by heating at 94° C. for 5 minutes; 5 cycles each consistingof reaction at 94° C. for 15 seconds and reaction at 72° C. for 3minutes; 5 cycles each consisting of reaction at 94° C. for 15 secondsand reaction at 72° C. for 3 minutes and 30 seconds; and 30 cycles eachconsisting of reaction at 94° C. for 15 seconds, reaction at 68° C. for30 seconds and reaction at 72° C. for 3 minutes, followed by reaction at72° C. for 10 minutes. The PCR was carried out using a PTC-200 DNAEngine (manufactured by Bio-Rad). The resulting PCR product of H chainand L chain had a size of about 700 bp and about 800 bp in the H chainand the L chain, respectively.

In order to determine the nucleotide sequence of the resulting PCRproduct, the PCR product was separated by agarose gel electrophoresisand extracted using a Gel Extraction Kit (manufactured by Qiagen). Theresulting extraction fragment was ligated into a pCR4TOPO vector using aTOPO TA cloning kit (manufactured by Invitrogen), and Escherichia coliDH5α (manufactured by Toyobo) was transformed with the resulting vector,using a method of Cohen et al [Proc. Natl. Acad. Sci. USA, 69, 2110(1972)]. A plasmid was extracted from the resulting transformant usingan automated plasmid isolation system PI-50 (manufactured by Kurabo) toobtain a plasmid, followed by reaction using a Big Dye Terminator CycleSequencing FS Ready Reaction Kit (manufactured by PE Biosystems) inaccordance with the instructions attached thereto and then thenucleotide sequence was analyzed using a DNA sequencer ABI PRISM 3700(manufactured by PE Biosystems).

As a result, a plasmid 08H2b10 comprising the full-length H chain cDNAof KM4008, a plasmid 08La4 comprising the full-length L chain cDNA ofKM4008, a plasmid 12Ha5 comprising the full-length H chain cDNA ofKM4012, a plasmid 12La4 comprising the full-length L chain cDNA ofKM4012, a plasmid 18rHa1 comprising the full-length H chain cDNA ofKM4018 and a plasmid 18Lb3 comprising the full-length L chain cDNA ofKM14018 in which an ATG sequence presumed to be an initiation codon waspresent at the 5′ terminal of cDNA were prepared.

A full-length nucleotide sequence of VH contained in the plasmid 08H2b10comprising the H chain cDNA of KM4008 was represented by SEQ ID NO:18, afull-length amino acid sequence of secretory VH comprising a signalsequence deduced from the full-length nucleotide sequence of VHcontained in the plasmid 08H2b10 was represented by SEQ ID NO:19, afull-length nucleotide sequence of VL contained in the plasmid 08La4comprising the L chain cDNA of KM4008 was represented by SEQ ID NO:20, afull-length amino acid sequence of secretory VL comprising a signalsequence deduced from the full-length nucleotide sequence of VLcontained in the plasmid 08La4 was represented by SEQ ID NO:21, afull-length nucleotide sequence of VH contained in the plasmid 12Ha5comprising the H chain cDNA of KM4012 was represented by SEQ ID NO:22, afull-length amino acid sequence of secretory VH comprising a signalsequence deduced from the full-length nucleotide sequence of VHcontained in the plasmid 12Ha5 was represented by SEQ ID NO:23, afull-length nucleotide sequence of VL contained in the plasmid 12La4comprising the L chain cDNA of KM4012 was represented by SEQ ID NO:24, afull-length amino acid sequence of secretory VL comprising a signalsequence deduced from the full-length nucleotide sequence of VLcontained in the plasmid 12La4 was represented by SEQ ID NO:25, afull-length nucleotide sequence of VH contained in the plasmid 18rHa1comprising the H chain cDNA of KM4018 was represented by SEQ ID NO:45, afull-length amino acid sequence of secretory VH comprising a signalsequence deduced from the full-length nucleotide sequence of VHcontained in the plasmid 18rHa1 was represented by SEQ ID NO:46, afull-length nucleotide sequence of VL contained in the plasmid 18Lb3comprising the L chain cDNA of KM4018 was represented by SEQ ID NO:47,and a full-length amino acid sequence of secretory VL comprising asignal sequence deduced from the full-length nucleotide sequence of VLcontained in the plasmid 18Lb3 was represented by SEQ ID NO:48,respectively.

(3) Analysis of Amino Acid Sequence of V Region of Anti-ASCT2 MonoclonalAntibody

The N-terminal amino acid sequences of the H chain and L chain in thepurified monoclonal antibodies of KM4008, KM4012 and KM4018 obtained inExample 5(7) were analyzed using a protein sequencer (PPSQ-10,manufactured by Shimadzu) and about 20 residues were determined. Fromthe comparison of the obtained analysis results with the amino acidsequences deduced from the nucleotide sequences of individual antibodiesobtained in (2), it was found that the individual sequences wereidentical to the corresponding sequence and thus confirmed that thenucleotide sequences obtained in the (2) were nucleotide sequences ofthe desired antibodies. Further, from the comparison with amino acidsequence data of known mouse antibodies or rat antibodies [SEQUENCES ofProteins of Immunological Interest, US Dept. Health and Human Services(1991)], it became clear that each of the isolated cDNAs was afull-length cDNA encoding the anti-ASCT2 monoclonal antibody KM4008,KM4012 or KM4018 comprising a secretory signal sequence; the secretorysignal sequence of the H chain and L chain of KM4008 were the amino acidsequence from positions 1 to 19 in the amino acid sequence representedby SEQ ID NO:19 and the amino acid sequence from positions 1 to 20 inthe amino acid sequence represented by SEQ ID NO:21, respectively; thesecretory signal sequence of the H chain and L chain of KM4012 were theamino acid sequence from positions 1 to 19 in the amino acid sequencerepresented by SEQ ID NO:23 and the amino acid sequence from positions 1to 20 in the amino acid sequence represented by SEQ ID NO:25,respectively; the secretory signal sequence of the H chain and L chainof KM4018 were the amino acid sequence from positions 1 to 19 in theamino acid sequence represented by SEQ ID NO:46 and the amino acidsequence from positions 1 to 20 in the amino acid sequence representedby SEQ ID NO:48, respectively.

Then, using the amino acid sequences of VH and VL of the anti-ASCT2monoclonal antibodies KM4008, KM4012 and KM4018, amino acid sequencedatabase of known proteins were searched by the BLASTP method [NucleicAcid Res. 25, 3389 (1997)]. As a result, no completely identical aminoacid sequence was found for both VH and VL and it was confirmed that VHand VL of the anti-ASCT2 monoclonal antibodies KM4008, KM4012, andKM4018 had novel amino acid sequences.

Further, the CDR sequences of VH and VL of the anti-ASCT2 monoclonalantibodies KM4008, KM4012, and KM4018 were identified by comparing themwith the amino acid sequences of known antibodies. Amino acid sequencesof CDR1, CDR2 and CDR3 of VH of the anti-ASCT2 monoclonal antibodyKM4008 were represented by SEQ ID NOs:26, 27 and 28, respectively andamino acid sequences of CDR1, CDR2 and CDR3 of VL thereof wererepresented by SEQ ID NOs:29, 30 and 31, respectively; amino acidsequences of CDR1, CDR2 and CDR3 of VH of the anti-ASCT2 monoclonalantibody KM4012 were represented by SEQ ID NOs:32, 33 and 34,respectively, and amino acid sequences of CDR1, CDR2 and CDR3 of VLthereof were represented by SEQ ID NOs:35, 36 and 37, respectively; andamino acid sequences of CDR1, CDR2 and CDR3 of VH of the anti-ASCT2monoclonal antibody KM4018 were represented by SEQ ID NOs:49, 50 and 51,respectively, and amino acid sequences of CDR1, CDR2 and CDR3 of VLthereof were represented by SEQ ID NOs:52, 53 and 54, respectively.

Example 8 Construction of Anti-ASCT2 Human Chimeric Antibody

(1) Construction of Anti-ASCT2 Human Chimeric Antibody-Expressing VectorcKM4008_(—)93

Using a vector pKANTEX93 (WO97/10354) for the expression of a humanizedantibody and a plasmid 08H2b 10 comprising an H chain cDNA of KM4008 anda plasmid 08La4 comprising an L chain cDNA of KM4008 each of whichobtained in Example 7(2), an anti-ASCT2 human chimericantibody-expressing vector cKM4008_(—)93 was constructed as follows.

The PCR was carried out in the same manner as in Example 2. To theplasmid 08H2b10 (100 ng) as a template, primers (10 pmol/L, each of 1μL) having the nucleotide sequences represented by SEQ ID NOs:38 and 39,10×Ex Taq buffer (5 μL), dNTPs (2.5 mmol/L, 4 μL), and Ex Taq polymerase(1 μL, all manufactured by Takara Bio) were added, followed by addingsterile water to give a total volume of 50 μL. The PCR was carried outby denaturation at 96° C. for 2 minutes; 30 cycles each consisting ofreaction at 94° C. for 1 minute, reaction at 55° C. for 1 minute, andreaction at 72° C. for 1 minute; and then reaction at 72° C. for 5minutes. As a result, a gene fragment encoding VH of KM4008 wasamplified in which a restriction enzyme recognizing sequence forinsertion into pKANTEX93 was added.

In addition, using the plasmid 08La4 (100 ng) as a template, primers (10μmol/L, each of 1 μL) having the nucleotide sequences represented by SEQID NOs:40 and 41, 10×Ex Taq buffer (5 μL), dNTPs (2.5 mmol/L, 4 μL) andEx Taq polymerase (1 μL, all manufactured by Takara Bio), the PCR wascarried out in the same manner as above to amplify a gene fragmentencoding VL of KM4008 in which a restriction enzyme recognizing sequencefor insertion into pKANTEX93 was added.

Each of the resulting reaction products was separated by agarose gelelectrophoresis and the about 0.5-kbp amplified fragment was extractedusing a Gel Extraction Kit (manufactured by Qiagen). The obtained genefragment was ligated into a pCR4TOPO vector using a TOPO TA cloning kit(manufactured by Invitrogen), and Escherichia coli DH5α (manufactured byToyobo) was transformed with the ligation product, as a same manner inExample 7(2), thereby obtaining a plasmid 08VH3 comprising a nucleotidesequence encoding VH of KM4008 and a plasmid 08VL6 comprising anucleotide sequence encoding VL of KM4008.

The resulting plasmid 08VH3 was digested with restriction enzymes ApaI(manufactured by Takara Bio) and Nod (manufactured by Takara Bio), andthe plasmid 08VL6 was digested with restriction enzymes EcoRI(manufactured by Takara Bio) and BsiWI (manufactured by Takara Bio) andseparated by agarose gel electrophoresis separation of the plasmids. Theresulting about 0.5-kbp gene fragment was extracted using a GelExtraction Kit (manufactured by Qiagen).

The resulting digested fragment of the plasmid 08VL6 was ligated intothe pKANTEX93 vector digested with the same restriction enzymes EcoRI(manufactured by Takara Bio) and BsiWJ (manufactured by Takara Bio).Escherichia coli DH5α (manufactured by Toyobo) was transformed with theligation product, in the same manner as Example 7(2), and the pKANTEX93vector in which VL of KM4008 were inserted was obtained using a plasmidisolation kit (manufactured by Qiagen).

The pKANTEX93 vector with insertion of VL of KM4008 was digested withApaI (manufactured by Takara Bio) and NotI (manufactured by Takara Bio)and then ligated into the above plasmid 08VH3-digested fragment. Then,Escherichia coli DH5α (manufactured by Toyobo) was transformed with theligation product, as a same manner in Example 7(2), and an anti-ASCT2human chimeric antibody expression vector cKM4008_(—)93 in which VH andVL of KM4008 were inserted was obtained using a plasmid isolation kit(manufactured by Qiagen).

(2) Construction of Anti-ASCT2 Human Chimeric Antibody-Expressing VectorcKM4012_(—)93

In the same manner as (1), an anti-ASCT2 human chimericantibody-expressing vector cKM4012_(—)93 was constructed from the vectorpKANTEX93 for the expression of a humanized antibody (WO97/10354), andthe plasmid 12Ha5 comprising the H chain cDNA of KM4012 and the plasmid12La4 comprising the L chain cDNA of KM4012 obtained in Example 7(2).

Using plasmids 12Ha5 and 12La4 as templates, the primers for theamplification of VH having the nucleotide sequences represented by SEQID NOs:38 and 42 and the primers for amplification of VL having thenucleotide sequences represented by SEQ ID NOs:40 and 41, the PCR wascarried out to amplify a gene fragment. Each of the resulting reactionproducts was separated, extracted and ligated into a pCR4TOPO vector.Escherichia coli DH5α (manufactured by Toyobo) was transformed with theligation product, thereby obtaining a plasmid 12VH1 comprising thenucleotide sequence encoding VH of KM4012, and a plasmid 12VL11comprising the nucleotide sequence encoding VL of KM4012.

The resulting plasmids 12VH1 and 12VL11 were digested with restrictionenzymes, respectively, separated and extracted to obtain fragments ofthe plasmid 12VH1 and the plasmid 12VL11. Each of the resultingfragments was ligated into the restriction enzyme-digested pKANTEX93vector. Then, Escherichia coli DH5α (manufactured by Toyobo) wastransformed with the ligation product and an anti-ASCT2 human chimericantibody expression vector cKM4012_(—)93 in which VH and VL of KM4012were inserted was obtained.

(3) Construction of Anti-ASCT2 Human Chimeric Antibody-Expressing VectorcKM4018_(—)93

In a same manner as the (1), an anti-ASCT2 human chimeric antibodyexpression vector cKM14018_(—)93 was constructed from the vectorpKANTEX93 for the expression of a humanized antibody (WO97/10354), andthe plasmid 18rHa1 comprising the H chain cDNA of KM4018 and the plasmid18Lb3 comprising the L chain cDNA of KM4018 obtained in Example 7(2).

Using the plasmids 18rHa1 and 18Lb3 as templates, the primers for theamplification of VH having the nucleotide sequences represented by SEQID NOs:55 and 56 and primers for the amplification of VL having thenucleotide sequences represented by SEQ ID NOs:57 and 58, PCR wascarried out to amplify the gene fragment. Each of the resulting reactionproducts was separated, extracted, and ligated into a pCR4TOPO vector,and Escherichia coli DH5α (manufactured by Toyobo) was transformed withthe ligation product, thereby obtaining a plasmid 18VH5 comprising anucleotide sequence encoding VH of KM4018, and a plasmid 18V2L1comprising a nucleotide sequence encoding VL of KM4018.

The resulting plasmids 18VH5 and 18V2L1 were digested with restrictionenzymes, respectively, separated, and extracted to obtain fragments ofthe plasmid 18VH5 and the plasmid 18V2L1. Each of the resultingfragments was ligated into the restriction enzyme-digested pKANTEX93vector. Then, Escherichia coli. DH5α (manufactured by Toyobo) wastransformed with the ligation product and an anti-ASCT2 human chimericantibody expression vector cKM14018_(—)93 was obtained in which VH andVL of KM4018 were instructed.

(4) Expression of Anti-ASCT2 Human Chimeric Antibody in Animal Cell

Using the anti-ASCT2 human chimeric antibody-expressing vectorcKM4008_(—)93 obtained in the above (1), expression of the anti-ASCT2human chimeric antibody in an animal cell was carried out by aconventional method [Antibody Engineering, A Practical Guide, W.H.Freeman and Company (1992)]. Similarly, using the anti-ASCT2 humanchimeric antibody-expressing vector cKM4012_(—)93, or the anti-ASCT2human chimeric antibody-expressing vector cKM4018_(—)93, expression ofindividual anti-ASCT2 human chimeric antibodies in an animal cell wascarried out. In this manner, transformants were obtained which producean anti-ASCT2 human chimeric antibodies.

(5) Obtaining of Purified Antibody

After each of transformants obtained in the above (4) was cultured by aconventional culturing method, the cell suspensions were collected andcentrifuged at 3,000 rpm and 4° C. for 15 minutes to recover thesupernatants. The culture supernatants were subjected to sterilefiltration using a 0.22-1 μm Millex GV filter (manufactured byMillipore). The anti-ASCT2 human chimeric antibodies cKM4008, cKM4012and cKM4018 (hereinafter referred to as cKM4008, cKM4012, and cKM4018,respectively) were purified from the resulting culture supernatants,using a Protein A High-capacity resin (manufactured by Millipore) columnin accordance with the instructions attached thereto.

The degree of purification and expressed molecular size of purifiedpreparations of the resulting cKM4008, cKM4012, cKM4018 were confirmedby SDS-PAGE using a gradient gel (Catalog Number: E-T520L, manufacturedby Atto) in accordance with the instructions attached thereto. Withregard to phoretic patterns of the purified anti-ASCT2 human chimericantibodies, one band was found around a molecular weight of 150 to 200kilodaltons (hereinafter, referred to as “kDa”) under non-reducingconditions, and two bands of about 50 kDa and about 25 kDa were foundunder reducing conditions [Antibodies-A Laboratory Manual, Cold SpringHarbor Laboratory, Chapter 14 (1988), Monoclonal Antibodies Principlesand Practice, Academic Press Limited (1996)]. Those phoretic patternswere consistent with the results for antibodies of the IgG class,obtained by SDS-PAGE under the same conditions. Thus, it was confirmedthat the anti-ASCT2 human chimeric antibodies cKM4008, cKM4012 andcKM4018 were expressed as antibody molecules having a correct structure.

Example 9 Investigation of Reactivity of Anti-ASCT2 Human ChimericAntibody

(1) Fluorescent Cell Staining Method (Flow Cytometer)

Each of the human ASCT2-myc/His gene-introduced CHO cell obtained inExample 2, and the multiple myeloma cell line OPM-2 (DSMZ Accession No.ACC50) was cultured in a 5% CO₂ incubator for 3 to 4 days at 37° C. Thehuman ASCT2-myc/His gene-introduced CHO cells were peeled off with a0.02% EDTA solution (manufactured by Nacalai Tesque), and in order toavoid the non-specific adsorption of antibodies, the cells were blockedfor 30 minutes at ice temperature using 1% BSA-PBS. The cells wereseeded into a 96-well U-bottom plate so as to give a density of 1×10⁵ to5×10⁵ cells/100 μL/well. The plates were centrifugation at 1,500 rpm for5 minutes (05PR-22, manufactured by Hitachi Koki), and then thesupernatant was removed. As the primary antibody, the purified antibodywhich was a test substance was diluted to give a final concentration of0.01 μg/mL to 10 μg/mL in 1% BSA-PBS, and dispensed to the plate at 100μL/well, followed by reaction at ice temperature for 60 minutes. Afterwashing the cells with 1% BSA-PBS, FITC-labeled anti-humanimmunoglobulin G (H+L) (manufactured by Jackson Laboratories) dilutedwith 1% BSA-PBS was added as the secondary antibody, followed byreaction at ice temperature for 30 minutes under shading. The cells werewashed with 1% BSA-PBS and then suspended in PBS. The fluorescenceintensity of the cells was measured by a flow cytometer (CytomicsFC500MPL, manufactured by Beckman Coulter).

The measurement results are given in FIG. 10. As shown in FIG. 10, eachof anti-ASCT2 human chimeric antibodies cKM4008, cKM4012 and cKM4018exhibited a potent binding activity for the human ASCT2-myc/Hisgene-introduced CHO cells. In addition, it was found that the chimericantibodies cKM4008, cKM4012 and cKM4018 exhibit a potent reactivity withthe multiple myeloma cell line OPM-2.

(2) ADCC Activity

The multiple myeloma cell line KMS-11 (HSRRB No. JCRB 1179) was used asa target cell. The cell was adjusted to give a cell density of 2×10⁵cells/mL using RPMI 1640 medium (manufactured by Invitrogen) containing5% FBS (manufactured by Invitrogen) and containing no Phenol Red(hereinafter, referred to as “ADCC activity assay medium”) and was usedas a target cell solution. Lymphoprep (manufactured by Nycomed) was usedfor the preparation of an effector cell solution, and, a peripheralblood mononuclear cell (PBMC) fraction was separated from healthy humanperipheral blood in accordance with the instructions attached thereto.The separated PBMC fraction was washed in an ADCC activity assay mediumby centrifugation twice, was adjusted to give a cell density of 2×10⁶cells/mL and then used as an effector cell solution.

Into a 96-well U-bottom plate (manufactured by Falcon), 50 μl, (1×10⁴cells/well) of the target cell solution was dispensed and then 50 μL ofthe effector cell solution (ratio of effector cells: target cells=10:1)was added. In addition, the anti-ASCT2 human chimeric antibody cKM4008,cKM4012 or cKM4018 was diluted in an ADCC activity assay medium. Thediluted antibody was added to the plate to give a total volume of 150 μLat a final concentration of 0.01 ng/mL to 1000 ng/mL, followed byreaction at 37° C. for 4 hours. After the reaction was complete, theplate was centrifuged and a lactic acid dehydrogenase (LDH) activity inthe supernatant was detected by measuring the absorbance using anLDH-Cytotoxic Test (manufactured by Wako Pure Chemical) in accordancewith the instructions attached thereto. The absorbance of spontaneousrelease of the target cells was obtained using an ADCC activity assaymedium in place of the effector cells solution and the antibodysolution, and the absorbance data of spontaneous release of the effectorcells was obtained using an ADCC activity assay medium in place of thetarget cell solution and the antibody solution, followed by carrying outthe same operation as above. The absorbance of total release of thetarget cells was obtained using an ADCC activity assay medium in placeof the antibody solution and the effector cell solution, by adding 20 μLof a 9% Triton X-100 solution at 45 minutes before completion of thereaction and carrying out the same operation as above. The ADCC activitywas calculated by the following formula.

(Formula)

ADCC activity (%)=[(absorbance of sample)−(absorbance of spontaneousrelease of effector cells/target cells)]/(absorbance of total release oftarget cells)-(absorbance of spontaneous release of target cells)]×100

The measurement results are shown in FIG. 11. As shown in FIG. 11, itwas demonstrated that the anti-ASCT2 human chimeric antibodies cKM4008,cKM4012 and cKM4018 exhibited an ADCC activity against the cells whichexpressed ASCT2 depending on an antibody concentration.

(3) CDC Activity

As target cells, the human ASCT2-myc/His gene-introduced CHO cellobtained in Example 2 and the colorectal cancer cell line Colo205 (ATCCAccession No. CCL-222) were peeled off using a 0.02%-EDTA Solution(manufactured by Nacalai Tesque), washed in RPMI 1640 medium(manufactured by Invitrogen) containing 1.4% BSA (manufactured byInvitrogen) and 50 μg/mL gentamicin (manufactured by Nacalai Tesque),which was prepared as a CDC assay medium and then suspended in the samemedium at a cell density of 2×10⁵ cells/mL. The resulting suspension wasused as a target cell solution.

The human complement serum (S2257-5ML, manufactured by Sigma) wasdissolved in 5 mL of deionized water and was two-fold diluted by addingan equal volume of a CDC assay medium. The resulting dilution was usedas a human complement solution.

Into a 96-well flat-bottom plate (manufactured by Sumitomo Bakelite), 50μL/well of the complement solution was dispensed. Then, to the well, 50μL of the target cell solution was added. Then, 50 μL of each ofantibody solutions diluted with a CDC assay medium was additionallyadded to give a total volume of 150 μL and reacted at 37° C. for 2 hoursin the presence of 5% CO₂. To the well, 15 μL/well of a WST-1 reagent(manufactured by Roche Diagnostics) was added. The resulting solutionwas stirred with a plate mixer, followed by at 37° C. for 2 hours in thepresence of 5% CO₂. The absorbance at 450 nm (control wavelength 650 nm)was measured using a microplate spectrophotometer (Emax microplatereader, manufactured by Molecular Devices). The absorbance of a well towhich 50 μl, of the complement solution and 100 μL of the CDC mediumwere added, was measured as blank. The absorbance of a well to which 50μL of the target cells, 50 μL of the complement solution and 50 μL ofthe CDC assay medium were added (antibody not added) were measured. TheCDC activity was calculated by the following formula.

(Formula)

CDC activity (%)={1-[(absorbance of antibody-added sample)-(absorbanceof blank)]/[(absorbance of non-antibody added sample)−(absorbance ofblank)]×100}

The measurement results are shown in FIG. 12. As shown in FIG. 12, itwas found that the anti-ASCT2 human chimeric antibodies cKM4008, cKM4012and cKM4018 exhibit a CDC activity against the cells which express ASCT2depending on the antibody concentration. In addition, it was also foundthat the anti-ASCT2 human chimeric antibodies cKM4008, cKM4012 andcKM4018 have a CDC activity against the colorectal cancer cell lineColo205.

(4) Inhibitory Activity on Intracellular Uptake of Amino Acids by ASCT2

Into a 96-well plate, 100 μL/well of the human colorectal cancer cellline WiDr (ATCC Accession No. CCL-218), which was adjusted to give acell density of 1×10⁴ cells/mL in a DMEM (manufactured by Invitrogen)containing 10% dFBS (manufactured by Invitrogen) (hereinafter, referredto as “glutamine-free medium”), was seeded and cultured in a 5% CO₂incubator at 37° C. for 24 hours.

To the well, 20 μL/well of each test substance, i.e., the anti-ASCT2human chimeric antibody, KM511 (negative control) or aglutamine-competitive amino acid mixture [AST mixture, a mixture ofalanine, serine and threonine (all manufactured by Sigma), 3.3 mmol/Lfor each)], which was diluted with PBS to give a final concentration of10 to 0.01 μg/mL, was added, followed by further addition of 20 μL/wellof a glutamine solution (manufactured by Invitrogen) which was preparedto give a final concentration of 0.2 mmol/L in a glutamine-free medium.To the wells of the control and the wells of the blank plate, 20 μL/wellof PBS and 20 μL/well of the above glutamine solution were added. Afteraddition of antibodies, the plates except for the blank plate wereincubated in a 5% CO₂ incubator at 37° C. for 72 hours.

Then, 20 μL/well of a WST-1 reagent (manufactured by Roche Diagnostics)diluted to 50% in the glutamine-free medium was added thereto andfurther incubated at 37° C. for 2 hours.

The absorbance at 450 nm (control wavelength 650 nm) was measured usinga microplate spectrophotometer (Emax microplate reader, manufactured byMolecular Devices). A relative proliferation rate (%) of the well withadding the antibody was calculated by regarding the absorbance of thewell of the comtrol without adding the antibody as 100% and regarding anabsorbance of the well of the blank plate as 0%.

The measurement results are shown in FIG. 13. As shown in FIG. 13, allof the anti-ASCT2 human chimeric antibodies cKM4008, cKM4012 and cKM4018remarkably inhibited cell proliferation depending on the antibodyconcentration. As a result, it was demonstrated that cKM4008, cKM4012and cKM4018 strongly neutralized the function of ASCT2 for intracellularuptake of glutamine, and consequently exhibited a significant inhibitoryactivity against the proliferation of cancer cells.

Example 10 Construction of Mouse ASCT2-Myc/his Gene-Introduced Cell Line(Hereinafter, Referred to as “Mouse ASCT2/CHO”)

In accordance with the following procedure, the plasmid pBluescript IISK (−)-Mouse_ASCT2-myc/His comprising the nucleotide sequencerepresented by SEQ ID NO:59 and the amino acid sequence represented bySEQ ID NO:60 was obtained, and a mouse ASCT2-myc/His gene-introduced CHOcell line was obtained using this plasmid.

Into 50 mL of an ampicillin-containing LB medium, 10 μL of the solutionof an E. coli comprising a mouse ASCT2 gene clone (Clone ID: 4192790,manufactured by Open Biosystems) was seeded, cultured under stirringovernight and then centrifuged (CR2DGII, manufactured by Hitachi Koki,6,000 rpm, 10 minutes) to recover bacteria. A plasmid comprising mouseASCT2 gene was prepared from the obtained bacteria using a plasmidisolation kit (manufactured by Qiagen). Total 100 μL of a solutioncontaining 100 ng of the obtained plasmid as a template, 10×KOD buffer I(10 μL), dNTPs (2 mmol/L, 5 μL), MgCl₂ (25 mmol/L, 4 ptL), primers (10μmol/L, each of 1 μL) having the nucleotide sequences represented by SEQID NOs:61 and 62, KOD polymerase (1 μL, manufactured by Toyobo), anddimethyl sulfoxide (5 μL) was prepared, and the PCR was carried out byreaction at 96° C. for 3 minutes; 35 cycles each consisting of reactionat 95° C. for 1 minute, reaction at 50° C. for 1 minute, and reaction at72° C. for 1.5 minutes; and then reaction at 72° C. for 7 minutes. Thereaction product was separated by agarose gel electrophoresis, and theresulting about 1.7-kb amplified fragment was extracted using a QIAquickGel Extraction Kit (manufactured by Qiagen). The resulting extractionfragment was digested with EcoRI (manufactured by Takara Bio) and KpnI(manufactured by Takara Bio) and then re-extracted using a QIAquick GelExtraction Kit. The extraction fragment was ligated into a pBluescriptII SK (−) vector digested with EcoRI (manufactured by Takara Bio) andKpnI (manufactured by Takara Bio) using a Ligation high (manufactured byToyobo), and then Escherichia coli DH5α (manufactured by Toyobo) wastransformed with the ligation product in accordance with the method ofCohen et al [Proc, Natl, Acad-Sci. USA. 69 2110 (1972)]. A plasmid wasextracted from the resulting transformant using an automated plasmidisolation system PI-50 (manufactured by Kurabo), and a plasmidpBluescript II SK (−)-Mouse_ASCT2-myc/His comprising the nucleotidesequence represented by SEQ ID NO:59 and the amino acid sequencerepresented by SEQ ID NO:60 was obtained.

The resulting pBluescript II SK (−)-Mouse_ASCT2-myc/His was digestedwith EcoRI (manufactured by Takara Bio) and KpnI (manufactured by TakaraBio), and a gene fragment was extracted in the same manner as in theabove and ligated into a pKANTEX93 vector (WO97/10354) which had beenpreviously digested with EcoRI and KpnI. Then, Escherichia coli DH5α(manufactured by Toyobo) was transformed with the ligation product inthe same manner as in the above, thereby obtaining a transformant. Theplasmid pKANTEx-Mouse_ASCT2-myc/His was obtained from the transformantusing a plasmid isolation kit (manufactured by Qiagen).

In accordance with the following procedure, the pKANTEX-MouseASCT2-myc/His was introduced into CHO/DG44 cells [Somatic cell andMolecular Genetics, 12, 555 (1986)] by an electroporation method[Cytotechnology, 3, 133 (1990)]. The cells used herein are thosesubcultured in a medium where 1×HT supplement (manufactured byInvitrogen) was added to IMDM (manufactured by Invitrogen) containing10% dFBS (manufactured by Invitrogen) and gentamicin (manufactured byNacalai Tesque, 50 μg/mL) (hereinafter, referred to as “A4 medium”). TheCHO/DG44 cells were suspended in buffer containing potassium chloride(137 nmol/L), sodium chloride (2.7 nmol/L), disodium hydrogen phosphate(8.1 mmol/L), sodium dihydrogen phosphate (1.5 nmol/L) and magnesiumchloride (4 mmol/L) (hereinafter, referred to as “K-PBS”) to give adensity of 8×10⁶ cells/mL, and the resulting cell suspension (200 μL,1.6×10⁶ cells in terms of cell count) was mixed with an expressionplasmid pKANTEX-Mouse_ASCT2-myc/His (10 μg). The mixture was transferredinto a cuvette (interelectrode distance: 2 mm), and the geneintroduction was carried out using a GenePulser II (manufactured byBio-Rad) at a pulse voltage of 0.35 kV and an electric capacity of 250μF. The cuvette was allowed to stand on ice, and a cell suspension inthe cuvette was suspended in a cell culture vessel containing an A4medium and cultured in a 5% CO₂ incubator at 37° C. After four dayculturing, the culture medium was exchanged with an A4 medium containing0.5 mg/mL of G418 (manufactured by Nacalai Tesque) and continued toculture the cells. With medium exchange and subculturing during the cellculture, a transformed cell line resistant to G418 was obtained abouttwo weeks after the gene introduction.

The resulting G418-resistant transformant cells were diluted to give acell density of 5 cells/mL in an A4 medium containing 0.5 mg/mL of G418(manufactured by Nacalai Tesque), and 100 μL/well of the diluted cellsuspension was dispensed into a 96-well plate, and treated with astepwise increasing concentration of methotrexate. In this manner, aclone which highly expresses mouse ASCT2-myc/His was selected, therebyobtaining mouse ASCT2/CHO.

Example 11 Construction of Mouse/Human Chimeric ASCT2-Myc/hisGene-Introduced Cell Line (Hereinafter, Referred to as “Mouse/HumanChimeric ASCT2/CHO”)

FIG. 14 shows a diagram of human and mouse ASCT2 proteins. In thediagram of the mouse ASCT2 protein in FIG. 14, the black portion (in anextracellular region of mouse ASCT2, amino acids corresponding topositions 74, 79, 84, 87, 90, 154, 159, 160, 163 to 171, 173, 174, 177,188, 204, 205, 207, 210 to 212, 214 to 223, 287, 293, 296, 297, 300, 301and 367 of human ASCT2) represents different amino acids between humanand mouse ASCT2. In the extracellular region of ASCT2, the number ofdifferent amino acids between human and mouse ASCT2 is 5 in EL1, so manyin EL2, 6 in EL3, and 1 in EL4. With regard to EL1, EL2 and EL3, inorder to cleave a domain containing each region at restriction enzymesites located near the domain and then obtain a mouse replacement formby recombination, the silent mutations were introduced using aQuickChange Site-Directed Mutagenesis Kit (manufactured by Stratagene)and thereby obtained a human ASCT2 gene in which an NcoI site was lostand a Ban/III site was introduced in a late region of EL3, and a mouseASCT2 gene in which an NcoI site in EL2 was lost. With regard to EL4which has one difference in the amino acid sequence, a human ASCT2 genewas converted into a mouse type by introducing mutation. A nucleotidesequence of the protein in which a myc/His tag was fused to theC-terminal side of each variant was constructed as follows, and thenintroduced into a CHO cell.

(1) Construction of variant human ASCT2 gene

PfuTubo DNA polymerase (1 μL) was added to total 50 μL of a solutioncontaining pCR4-SLC1A5-myc/His constructed in Example 2 as a template,the primers (0.1 μg/mL, 2.5 μL) comprising the nucleotide sequencerepresented by SEQ ID NOs:63 and 64, 10× reaction buffer (5 μL) attachedto QuikChange Site-Directed Mutagenesis Kit and dNTPs (1 μL), and PCRwas carried out by reaction at 95° C. for 30 seconds; 18 cycles eachconsisting of three processes, reaction at 95° C. for 30 seconds,reaction at 55° C. for 1 minute, and reaction at 68° C. for 5 minute;and then reaction at 37° C. for 1 hour with addition of 1 μL of DnpIafter completion of the reaction. Escherichia coli XL 1-Blue(manufactured by Stratagene) attached to a QuickChange Site-DirectedMutagenesis Kit was transformed with 2 μL of the reaction product. Aplasmid was extracted from the resulting transformant using an automatedplasmid isolation system PI-50 (manufactured by Kurabo) and a plasmidpCR4-hNcoIKO_ASCT2-myc/His was obtained. Using this plasmid as atemplate, and the primers (0.1 μg/mL, 2.5 μL) comprising the nucleotidesequences represented by SEQ ID NOs:65 and 66, respectively, a plasmidpCR4-hBam3KI_ASCT2-myc/His was obtained in the same manner as in theabove. The obtained plasmid was digested with EcoRI (manufactured byTakara Bio) and KpnI (manufactured by Takara Bio), extracted using aQIAquick Gel Extraction Kit, and was ligated into a pBluescript II SK(−) vector (manufactured by Stratagene) which had been previouslydigested with EcoRI and KpnI, using a Ligation high (manufactured byToyobo). Then, Escherichia colit DH5α (manufactured by Toyobo) wastransformed with the ligation product, in accordance with the method ofCohen et al [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], and a plasmidpBluescript II SK (−) hBam3KI_ASCT2-myc/His was obtained in the samemanner as above.

(2) Construction of Variant Mouse ASCT2 Gene

Using pBluescript II SK (−)-Mouse-ASCT2-myc/His constructed in Example10 as a template, and primers comprising the nucleotide sequencesrepresented by SEQ ID NOs:67 and 68, respectively, a plasmid pBluescriptII SK (−)-mNcoKO_ASCT2-myc/His was obtained in the same manner as inExample 11(1).

(3) Construction of Mouse/Human Chimeric ASCT2/CHO

A plasmid, which was obtained by re-transforming dam-Escherichia coli(Catalog Number: C2925, manufactured by New England Biolabs) with theplasmids obtained in (1) and (2), was digested with EcoR1 (manufacturedby Takara Bio) and Bell (equivalent to FbaI, manufactured by TakaraBio), and ligated into a plasmid comprising a human ASCT2 gene as avector, using a mouse ASCT2 gene as an insert. Then, Escherichia coliDH5α (manufactured by Toyobo) was transformed with the ligation productto obtain a plasmid pBluescript II SK (−)-mEL1 ASCT2-myc/His in whichEL1 was replaced with a mouse type of ELL Similarly, a plasmidpBluescript II SK (−)-mEL2 ASCT2-myc/His and a plasmid pBluescript II SK(−)-mEL3 ASCT2-myc/His were obtained by replacing EL2 with a mouse typeof EL2 at Bell and NcoI sites and replacing EL3 with a mouse type of EL3at NcoI and BamIII sites, respectively.

Using the pBluescript II SK (−)hBam3KI_ASCT2-myc/His obtained in (1) asa template, and the primers having the nucleotide sequences representedby SEQ ID NOs:69 and 70, the plasmid pBluescript II SK(−)-mEL4_ASCT2-myc/His in which EL4 was replaced with a mouse type ofEL4 was obtained in the same manner as in Example 11(1).

The resulting pBluescript II SK (−)-mEL1_ASCT2-myc/His, pBluescript IISK (−)-mEL2_ASCT2-myc/His, pBluescript II SK (−)-mEL3_ASCT2-myc/His andpBluescript II SK (−)-mEL4-ASCT2-myc/His were digested with EcoRI(manufactured by Takara Bio) and KpnI (manufactured by Takara Bio) andeach of gene fragments was extracted in the same manner as above, andligated into a pKANTEX93 vector (WO97/10354) which had been previouslydigested with EcoRI and KpnI. Then, Escherichia coli DH5α (manufacturedby Toyobo) was transformed with the ligation product to obtaintransformants. Then, the expression plasmids pKANTEX-mEL1_ASCT2-myc/His,pKANTEX-mEL2_ASCT2-myc/His, pKANTEX-mEL3_ASCT2-myc/His andpKANTEX-mEL4_ASCT2-myc/His were obtained from the respectivetransformants using a plasmid isolation kit (manufactured by Qiagen).

In accordance with the electroporation method,pKANTEX-mEL1-ASCT2-myc/His, pKANTEX-mEL2_ASCT2-myc/His,pKANTEX-mEL3_ASCT2_myc/His and pKANTEX-mEL4_ASCT2-myc/His wereintroduced into CHO/DG44 cells respectively in the same manner as inExample 10, and thereby obtained a mouse EL1 type ASCT2-myc/Hisgene-introduced CHO cell line (hereinafter, referred to as “mEL1/CHO”),a mouse EL2 type ASCT2-myc/His gene-introduced CHO cell line(hereinafter, referred to as “mEL2/CHO”), a mouse EL3 type ASCT2-myc/Hisgene-introduced CHO cell line (hereinafter, referred to as “mEL3/CHO”)and a mouse EL4 type ASCT2-myc/His gene-introduced CHO cell line(hereinafter, referred to as “mEL4/CHO”), respectively.

Example 12 Reactivity of Anti-ASCT2 Monoclonal Antibody with MouseASCT2/CHO and Mouse/Human Chimeric ASCT2/CHO

Using the mouse ASCT2/CHO and mouse/human chimeric ASCT2/CHO cellsconstructed in Examples 10 and 11 and FITC-labeled anti-mouseimmunoglobulin G (H+L) (manufactured by Dako) or ALEXA488-labeledanti-rat immunoglobulin G (G+L) (manufactured by Invitrogen) as thesecondary antibody, the reactivity of anti-ASCT2 monoclonal antibodiesKM4008, KM4012 and KM4018 was measured in the same manner as in Example9(1).

The measurement results are shown in FIG. 15. As shown in FIG. 15, allof the anti-ASCT2 monoclonal antibodies KM4008, KM4012 and KM4018exhibited no reactivity with mouse ASCT2. In addition, KM4008 and KM4012exhibited a decreased reactivity with mEL2/CHO. On the other hand,KM4018 exhibited no reactivity with mEL2/CHO, and a decreased reactivitywith mEL1/CHO and mEL3/CHO.

From these results, it was demonstrated that the anti-ASCT2 monoclonalantibodies KM4008 and KM4012 recognize and bind to EL2, and theanti-ASCT2 monoclonal antibody KM4018 recognizes and binds to athree-dimensional structure comprising EL1, EL2 and EL3.

Example 13 Preparation of Anti-ASCT2 Humanized Antibody

(1) Design of Amino Acid Sequences of VH and VL of Anti-ASCT2 HumanizedAntibody

An amino acid sequence of VH of an anti-ASCT2 humanized antibody wasdesigned as follows.

Firstly, the amino acid sequence of FR of VH of a human antibody wasselected in order to graft amino acid sequences of CDR1 to CDR3 ofKM4008VH represented by SEQ ID NOs:26 to 28, respectively, obtained inExample 7(3). Using a GCG Package (manufactured by Genetics ComputerGroup) as a sequence analysis system, based on the amino acid sequencedatabase of conventional proteins by the BLASTP method [Nucleic AcidsRes., 25, 3389 (1997)], a human antibody having a high homology withKM4008 was searched. When the homology score was compared with thehomology of an actual amino acid sequence, SWISSPROT database accessionno. BAC01342, the immunoglobulin heavy chain VHDJ region (hereinafter,referred to as “BAC01342”) exhibited a homology of 77.0%, and it was ahuman antibody which had the highest homology, so the amino acidsequence of FR of this antibody was selected.

Amino acid sequences of CDRs of VH of KM4008 represented by SEQ IDNOs:26 to 28 were grafted into an appropriate position of the thusdetermined amino acid sequence of FR of the human antibody. In thismanner, an amino acid sequence HV0 of VH of the anti-ASCT2 humanizedantibody represented by SEQ ID NO:71 was designed.

Next, an amino acid sequence of VL of the anti-ASCT2 humanized antibodywas designed as follows.

An amino acid sequence of FR of VL of a human antibody was selected inorder to graft amino acid sequences of CDR1 to CDR3 of KM4008VLrepresented by SEQ ID NOs:29 to 31, respectively, obtained in Example7(3). Kabat et al. have classified VL of conventionally known varioushuman antibodies into four subgroups (HSG I to IV) based on the homologyof their amino acid sequences and reported on the consensus sequencesfor each of the subgroups [Sequences of Proteins of ImmunologicalInterest, US Dept. Health and Human Services (1991)]. Accordingly, thehomology was examined between the amino acid sequences of FR ofconsensus sequences of subgroups I to IV of VL of the human antibody andthe amino acid sequence of FR of VL of KM4008.

As a result of the homology analysis, the homology of HSG I, HSG II, HSGIII, and HSG IV was 77.5%, 60.0%, 63.8%, and 68.8%, respectively.Therefore, the amino acid sequence of FR of KM4008VL had the highesthomology with the subgroup I.

Based on these results, the amino acid sequence of CDR of VL of KM4008was grafted into an appropriate position of an amino acid sequence of FRof the consensus sequence of subgroup I of VL of the human antibody.However, since Leu at position 124 in the amino acid sequence of VL ofKM4008 represented by SEQ ID NO:21 was not the amino acid residue havingthe highest use frequency in the region which corresponded to the aminoacid sequence of the human antibody FR cited by Kabat, but is an aminoacid residue which was used at a relatively high frequency, the aboveamino acid residue recognized in the amino acid sequence of KM4008 wasused. In this manner, the amino acid sequence LV0 of VL of an anti-ASCT2humanized antibody represented by SEQ ID NO:72 was designed.

The amino acid sequence HV0 of VH and amino acid sequence LV0 of VL ofthe anti-ASCT2 humanized antibody designed in the above are sequences inwhich the amino acid sequence of CDR of the anti-ASCT2 mouse monoclonalantibody KM4008 alone was grafted into the selected amino acid sequenceof FR of the human antibody. However, it is known that when a humanizedantibody is produced by simply grafting only CDRs of a mouse antibodyinto FRs of a human antibody, its antigen-binding activity is lower thanthat of the original mouse antibody. For these reasons, in order toavoid lowering of the binding activity, the amino acid residues whichare considered to have an influence on the binding activity, among theamino acid residues of FRs which are different between human antibodiesand mouse antibodies, are usually modified together with the grafting ofthe amino acid sequence of CDRs. Thus, in this Example, the amino acidresidues of FRs which is considered to influence on the binding activitywere identified as follows.

Firstly, a three-dimensional structure of V region of the antibody(hereinafter, referred to as “HV0LV0”) comprising the amino acidsequence of HV0 of VH and amino acid sequence of LV0 of VL of anti-ASCT2humanized antibody designed in the above was constructed using acomputer modeling technique. The three dimensional structure wasprepared and displayed using Discovery Studio (manufactured by Accelrys)in accordance with instructions attached thereto. In addition, acomputer model of the three-dimensional structure of the V region of theanti-ASCT2 mouse monoclonal antibody KM4008 was also constructed in thesame manner. Further, in the amino acid sequence of FRs of VH and VL ofHV0LV0, amino acid residues which were different from those of KM4008were selected and replaced with the corresponding amino acid residues ofKM4008, and the three-dimensional structure model was constructed basedon the resulting amino acid sequence in the same manner. The amino acidresidues predicted to influence on the binding activity of the antibodywere identified by comparing three-dimensional structures of the Vregions of the constructed KM4008, HV0LV0 and modified product.

As a result, as the amino acid residues among amino acid residues of FRof HV0LV0, which are considered to change a three-dimensional structureof the antigen binding region and therefore influence on the bindingactivity of the antibody, Val at position 2, Ser at position 9, Val atposition 20, Ser at position 30, Arg at position 38, Glu at position 46,Leu at position 86, Val at position 93, Tyr at position 95 and Val atposition 116 in the HV0 sequence, and Pro at position 8, Val at position15, Gln at position 38, Ala at position 43, Pro at position 44, Phe atposition 71, and Tyr at position 87 in the LV0 sequence wererespectively selected.

By modifying at least one or more amino acid sequences of these selectedamino acid residues into the amino acid residues which are present atthe same positions of KM4008, VH and VL of a humanized antibody havingvarious modifications were designed.

Specifically, regarding VH, at least one modification among the aminoacid modifications for substituting Val at position 2 with Ile, Ser atposition 9 with Pro, Val at position 20 with Ile, Ser at position 30with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leuat position 86 with Val, Val at position 93 with Thr, Tyr at position 95with Phe, and Val at position 116 with Leu was introduced in the aminoacid sequence represented by SEQ ID NO:71. Further, regarding VL, atleast one modification among the amino acid modifications forsubstituting Pro at position 8 with Thr, Val at position 15 with Leu,Gln at position 38 with Arg, Ala at position 43 with Thr, Pro atposition 44 with Val, Phe at position 71 with Tyr, and Tyr at position87 with Phe was introduced in the amino acid sequence represented by SEQID NO:72.

As a result, HV0LV3, HV2LV0, HV2LV3, HV4LV0, HV4LV3, HV7LV0, HV7LV3,HV10LV0, and HV10LV3 were respectively designed as antibody V regions ofthe anti-ASCT2 humanized antibody with modifications of at least one ofamino acid residues present in FR of HV0LV0. The amino acid sequences ofH chain variable regions HV2, HV4, HV7 and HV 10, and L chain variableregion LV3 were represented by SEQ ID NOs:76, 78, 80, 82 and 84,respectively.

(2) Preparation of Anti-ASCT2 Humanized Antibody

A DNA encoding the amino acid sequence of the variable region of theanti-ASCT2 humanized antibody was prepared in mammalian cells using acodon which was used at a high frequency, when amino acidmodification(s) were carried out using a codon which is used as a DNAencoding the amino acid sequence of VH or VL of KM4008. The nucleotidesequences encoding the amino acid sequence of HV0 and LV0 of theanti-ASCT2 humanized antibody were represented by SEQ ID NOs:73 and 74,respectively, whereas the nucleotide sequences encoding the amino acidsequences of variable regions HV2, HV4, HV7, HV10 and LV3 on which aminoacid modification(s) were made were represented by SEQ ID NOs:75, 77,79, 81 and 83, respectively.

Using these nucleotide sequences, humanized antibody-expressing vectorswere constructed and humanized antibodies were respectively expressed inthe same manner as in Example 8.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skill in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

This application is based on Japanese application No. 2008-185790, filedon Jul. 17, 2008, and U.S. provisional application No. 61/081,820, filedon Jul. 18, 2008, the entire contents of which are incorporated hereintoby reference. All references cited herein are incorporated in theirentirety.

1-28. (canceled)
 29. A DNA encoding a monoclonal antibody or fragmentthereof which specifically binds to a native three-dimensional structureof an extracellular region of system ASC amino acid transporter 2(ASCT2).
 30. A recombinant vector comprising the DNA of claim
 29. 31. Atransformant obtainable by introducing the recombinant vector of claim30 into a host cell.
 32. A therapeutic or diagnostic method for treatingor diagnosing a disease relating to ASCT2, comprising using a monoclonalantibody or fragment thereof which specifically binds to a nativethree-dimensional structure of an extracellular region of system ASCamino acid transporter 2 (ASCT2).
 33. The therapeutic or diagnosticmethod according to claim 32, wherein the disease relating to ASCT2 iscancer.
 34. The therapeutic or diagnostic method according to claim 33,wherein the cancer is selected from the group consisting of bloodcancer, esophageal cancer, gastric cancer, colorectal cancer, livercancer or prostate cancer.
 35. A method for immunologically detecting ormeasuring ASCT2 or a ASCT2-expressing cell, comprising using amonoclonal antibody or fragment thereof which specifically binds to anative three-dimensional structure of an extracellular region of systemASC amino acid transporter 2 (ASCT2).
 36. The method according to claim35, wherein the method is an immunoprecipitation method and/or afluorescent cell staining method.